Vault complexes for cytokine delivery

ABSTRACT

The invention relates to compositions of vault complexes containing recombinant cytokine fusion proteins that include a cytokine and a vault targeting domain, and methods of using the vault complexes to deliver the cytokines to a cell or subject, and methods for using the compositions to treat cancer, such as lung cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/257,358, filed Nov. 2, 2009, the entire disclosure of which is herebyincorporated by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.5R01CA126944, awarded by the National Institutes of Health and wassupported by the U.S. Department of Veterans Affairs. The Government hascertain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 11111US_sequencelisting.txt, created on Month XX,201X, with a size of XXX,XXX bytes. The sequence listing is incorporatedby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to compositions of vault complexes containingcytokines, such as the chemokine CCL-21, and use of vault complexes fordelivering the cytokines to a cell. The vault complexes include a fusionprotein of the cytokine of interest fused to major vault interactiondomain. Also included in the invention is the use of the compositions ascancer immunotherapy agents for activating an immune response against atumor and for treating cancers, including lung cancer.

2. Description of the Related Art

Vaults are cytoplasmic ubiquitous ribonucleoprotein particles firstdescribed in 1986 that are found in all eukaryotic cells [1]. Nativevaults are 12.9±1 MDa ovoid spheres with overall dimensions ofapproximately 40 nm in width and 70 nm in length [2,3], present innearly all-eukaryotic organisms with between 10⁴ and 10⁷ particles percell [4]. Despite their cellular abundance, vault function remainselusive although they have been linked to many cellular processes,including the innate immune response, multidrug resistance in cancercells, multifaceted signaling pathways, and intracellular transport [5].

Vaults are highly stable structures in vitro, and a number of studiesindicate that the particles are non-immunogenic [6]. Vaults can beengineered and expressed using a baculovirus expression system andheterologous proteins can be encapsulated inside of these recombinantparticles using a protein-targeting domain termed INT for vaultINTeraction. Several heterologous proteins have been fused to the INTdomain (e.g. fluorescent and enzymatic proteins) and these fusionproteins are expressed in the recombinant vaults and retain their nativecharacteristics, thus conferring new properties onto these vaults [7,8].

CCL-21 has been identified as a lymphoid chemokine that is predominantlyand constitutively expressed by high endothelial venules in lymph nodesand Peyer's patches and by lymphatic vessels, stromal cells in thespleen and appendix [9]. CCL-21 binds to the chemokine receptor CCR7 andis a chemoattractant for mature DCs, naive and memory T cells [10,11].This chemokine, along with CCL-19, is required for normal lymphoidtissue organization that is ultimately essential for effective T cell-DCinteractions. Natural killer (NK) and natural killer T (NKT) antitumoreffectors express the CCR7 receptor and are chemo attracted by CCL-21.The use of chemokines that attract DC, lymphocyte, and NK and NKTeffectors into tumors can serve as an effective antitumor strategy.Based on this concept, it has been previously shown that intratumoraladministration of recombinant CCL-21 reduces tumor burden in murine lungcancer models [12]. The antitumor responses induced by recombinantCCL-21 however required high and frequent dosing because proteinsadministered intratumorally are not retained locally for prolongedperiods. Although these studies delineated the role of CCL-21 as aneffective antitumor agent, frequent high dose intratumoraladministration is clinically limiting with the potential of unnecessarysystemic toxicity. Based on the limitations of this approach, the use ofautologous dendritic cells for intratumoral CCL-21 delivery was examined[13.14]. In preclinical studies, it was demonstrated that intratumoraladministration of CCL-21 gene modified dendritic cells led to tumoreradication in murine lung cancer models. Following this initialdescription of the antitumor properties of CCL-21, several otherresearch groups have reported that CCL-21 has potent antitumorproperties in a variety of model systems [15-19]. In all models, CCL-21demonstrated potent regression of tumors, which was shown to bedependent on host T cell immunity. Based on extensive pre-clinicalevaluation, the intratumoral injection of DC transduced with anadenoviral vector expressing the secondary lymphoid chemokine gene(Ad-CCL-21-DC) was assessed in a phase I trial in advanced non-smallcell lung cancer (NSCLC).

While clinical studies utilizing intratumoral administration ofchemokine gene modified DC show promise as an effective therapy, thepreparation of CCL-21 expressing autologous dendritic cells iscumbersome, expensive and time consuming. A reagent that is efficaciousand works through a similar therapeutic mechanism is highly desired.Compositions and methods are needed to circumvent autologous DCpreparation, minimize batch to batch variability and allow forcomparability and standardization. There is a need for cytokinedelivery, e.g., a non-DC based approach for intratumoral CCL-21 deliveryfor the purpose of initiating antitumor immune responses

Vaults are generally described in U.S. Pat. No. 7,482,319, filed on Mar.10, 2004; U.S. application Ser. No. 12/252,200, filed on Oct. 15, 2008;International Application No. PCT/US2004/007434, filed on Mar. 10, 2004;U.S. Provisional Application No. 60/453,800, filed on Mar. 20, 2003;U.S. Pat. No. 6,156,879, filed on Jun. 3, 1998; U.S. Pat. No. 6,555,347,filed on Jun. 28, 2000; U.S. Pat. No. 6,110,740, filed on Mar. 26, 1999;International Application No. PCT/US1999/06683, filed on Mar. 26, 1999;U.S. Provisional App. No. 60/079,634, filed on Mar. 27, 1998; andInternational Application No. PCT/US1998/011348, filed on Jun. 3, 1998.Vault compositions for immunization against chlamydia genital infectionare described in U.S. application Ser. No. 12/467,255, filed on May 15,2009. The entire contents of these applications are incorporated byreference in their entirety for all purposes.

SUMMARY OF THE INVENTION

Disclosed herein are compositions including a vault complex having afusion protein of a cytokine and a vault targeting domain, e.g., mINT.In one embodiment, the vault complex includes a chemokine fusion proteinhaving a chemokine (C-C motif) ligand 21 (CCL-21) consisting of SEQ IDNO:1 (mouse CCL21 amino acid sequence) and a major vault proteininteraction domain (mINT) consisting of SEQ ID NO:9 (mouse mINT aminoacid sequence). In another embodiment, the vault complex includes achemokine fusion protein having a chemokine (C-C motif) ligand 21(CCL-21) consisting of SEQ ID NO:2 (human CCL21 amino acid sequence) anda major vault protein interaction domain (mINT) consisting of SEQ IDNO:8 (human mINT amino acid sequence).

Accordingly, in one aspect of the invention, the cytokine is achemokine. In one aspect, the cytokine is a cysteine-cysteine (CC)chemokine. In another aspect, the cytokine is a CCL-21 chemokine. Thechemokine can include all or part of human or mouse CCL-21, e.g, SEQ IDNO:1 or SEQ ID NO:2. In some embodiments, the cytokine fusion proteinincludes a fluorescent protein, e.g., mCherry fluorescent protein.

In one embodiment, the vault targeting domain is a vault interactiondomain from a vault poly ADP-ribose polymerase (VPARP). In oneembodiment, the vault targeting domain is a major vault proteininteraction (mINT) domain. In another embodiment, the vault targetingdomain comprises or consists of SEQ ID NO:8 (a human amino acidsequence). In yet another embodiment, the vault targeting domaincomprises SEQ ID NO:9 (a mouse amino acid sequence).

In some embodiments, the vault complex includes a MVP. The MVP can be ahuman MVP, e.g., SEQ ID NO:16.

Vault complexes of the invention can include a vault poly ADP-ribosepolymerase (VPARP), and/or a telomerase vault associated protein 1(TEP1), and/or an untranslated RNA molecule (vRNA).

In addition, the invention provides an isolated nucleic acid encoding acytokine fusion protein that includes a cytokine encoding sequence and amINT encoding sequence. In one embodiment, the mINT encoding sequence isSEQ ID NO:7 (a human sequence) or SEQ ID NO:6 (a mouse sequence). Inanother embodiment, the cytokine encoding sequence is SEQ ID NO:5(human) and the mINT encoding sequence consists of SEQ ID NO:7 (human).In one embodiment, the cytokine encoding sequence is SEQ ID NO:3 (amouse sequence) and the mINT encoding sequence is SEQ ID NO:6 (a mousesequence).

In some embodiments, the cytokine fusion protein is SEQ ID NO:13(human). In other embodiments, the cytokine fusion protein is SEQ IDNO:12 (mouse). Also included in the invention are vectors including anisolated nucleic acid described herein, cells having an isolated nucleicacid described herein, and cells having a vector described herein.

The invention also includes a method of delivering a cytokine to a cell,including introducing the vault complexes of the invention to the cell.In some embodiments, the method includes introducing the vault complexesinto the extracellular environment surrounding the cell. The inventionincludes a method for stimulating an immune response in a cell bycontacting the cell with the vault complexes of the invention. In someembodiments, the cell is a human cell. In other embodiments, the immuneresponse induces migration of T cells and dendritic cells. In anotherembodiment, contacting the cell with the vault complexes of theinvention increases T cell migration to the cell by at least 5% comparedto administration of CCL-21 cytokine alone.

In addition, the invention provides a method for stimulating an immuneresponse in a subject by administering the vault complexes the inventionto the subject. In one embodiment, the subject is a human.

In another embodiment, the invention includes a method of treating ormanaging cancer in a subject in need of treatment or management ofcancer including administering to a subject a therapeutically effectiveamount of the vault complexes described herein. In some embodiments,administering includes intra-tumoral injection of the composition to atumor in the subject. In one embodiment, the cancer is lung cancer. Inanother embodiment, administering reduces tumor volume and/or reducestumor growth. In some embodiments, administering increases interleukin-2(IL-2) expression. In one embodiment, the method includes a subject thatis a mammal or a human.

The invention includes a method of preparing the vault complexes of theinvention including a) mixing a fusion protein comprising a cytokinefused to a mINT generated in Sf9 cells with a rat MVP generated in Sf9cells to generate a mixture; b) incubating the mixture for a sufficientperiod of time to allow packaging of the fusion protein inside of vaultcomplexes, thereby generating the vault complexes.

In yet another embodiment, the invention also provides method ofpreparing the vault complexes of the invention including a) mixing afusion protein comprising a cytokine fused to a mINT generated in insectlarvae cells with a rat MVP generated in insect larvae cells to generatea mixture; b) incubating the mixture for a sufficient period of time toallow packaging of the fusion protein inside of vault complexes, therebygenerating the vault complexes described herein.

In another embodiment, the invention provides a method of preparing thecomposition of the invention including a) mixing a fusion proteincomprising a cytokine fused to a mINT generated in Sf9 cells or insectlarvae cells with a human MVP generated in Sf9 cells or insect larvaecells to generate a mixture; b) incubating the mixture for a sufficientperiod of time to allow packaging of the fusion protein inside of vaultcomplexes, thereby generating the vault complexes described herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1A shows a diagram of the CCL-21 and mCherry-INT constructs thatwere fused to create the CCL-21 fusion protein.

FIG. 1B shows the incorporation into a pFastBac expression vector byrestriction digest and expression of CCL-21 fusion protein as analyzedby gel electrophoresis.

FIG. 1C shows the MVP recombinant vaults containing packagedCCL-21-mCherry-mINT purified on a sucrose gradient. The 40 and 45%fractions were analyzed by SDS-PAGE.

FIG. 1D shows the MVP recombinant vaults containing packagedCCL-21-mCherry-mINT purified on a sucrose gradient and analyzed bystaining with Coomassie.

FIG. 1E shows purified vault complexes examined by negative staintransmission electron microscopy.

FIG. 2A is a graph that shows CCL-21 vault complexes increases themigration of T2 cells.

FIG. 2B is a graph showing T cell activation by CCL-21 vault complexesmeasured by IL-2 production.

FIG. 3 is a graph showing a decrease in tumor burden by intratumoralinjection of CCL-21 vault complexes (200 ng) compared to empty vaults.

FIG. 4A shows the leukocytic infiltrates in cells treated with a controlvault.

FIG. 4B shows the leukocytic infiltrates in cells treated with CCL-21vault complexes.

FIG. 5A is a photo showing the effects of treatment of 3LL lung cancercells with diluent.

FIG. 5B is a photo showing the effects of treatment of 3LL lung cancercells with control vault.

FIG. 5C is a photo showing the effects of treatment of 3LL lung cancercells with CCL-21 vault complex.

FIG. 6A is a set of graphs showing the percentage tumor burden in naïve3LL cells, and 3LL cells treated with diluents, control vault (VC), orCCL-21 vault complexes as measured in a flow cytometry assay. Tumorburden was calculated on total percentage of GFP and Epcam expressingtumor cells in total lung digest.

FIG. 6B is a bar graph showing percentage tumor burden in 3LL cellsafter treatment with diluents, control vault, or CCL-21 vault complexes.

FIG. 7A is a graph showing percentage of intratumoral leukocyticpopulations (CD4, CD8, CD3 CXCR3, CD3 CCR7, DEC205, MDSC and Tregs) atthe tumor site after injection with diluent, control vault, or CCL-21vault complexes. CCL-21 vault complex augmented CD4, CD8, CXCR3⁺CD3⁺T,CCR7⁺CD3⁺T and DEC205⁺DC infiltrates and reduced MDSC and Tregs.

FIG. 7B is a graph of the percentage of CD4+ cells with IFNγ and IL-10expression after treatment with diluent, control vault, or CCL-21 vaultcomplexes. Tumor T lymphocytic infiltrates from CCL-21 vault complextreated mice had increased intracytoplasmic IFNγ and reduced IL-10expression.

FIG. 7C is a graph of the percentage of CD8+ cells with IFNγ and IL-10expression after treatment with diluent, control vault, or CCL-21 vaultcomplexes.

FIG. 7D is a graph of percentage tumor lysis of splenic T cells afterintratumoral injection with diluent, control vault, or CCL-21 vaultcomplexes.

DETAILED DESCRIPTION OF THE INVENTION

The descriptions of various aspects of the invention are presented forpurposes of illustration, and are not intended to be exhaustive or tolimit the invention to the forms disclosed. Persons skilled in therelevant art can appreciate that many modifications and variations arepossible in light of the embodiment teachings.

It should be noted that the language used herein has been principallyselected for readability and instructional purposes, and it may not havebeen selected to delineate or circumscribe the inventive subject matter.Accordingly, the disclosure is intended to be illustrative, but notlimiting, of the scope of invention.

It must be noted that, as used in the specification, the singular forms“a”, “an”, and “the” include plural referents unless the context clearlydictates otherwise.

Any terms not directly defined herein shall be understood to have themeanings commonly associated with them as understood within the art ofthe invention. Certain terms are discussed herein to provide additionalguidance to the practitioner in describing the compositions, devices,methods and the like of embodiments of the invention, and how to make oruse them. It will be appreciated that the same thing can be said in morethan one way. Consequently, alternative language and synonyms can beused for any one or more of the terms discussed herein. No significanceis to be placed upon whether or not a term is elaborated or discussedherein. Some synonyms or substitutable methods, materials and the likeare provided. Recital of one or a few synonyms or equivalents does notexclude use of other synonyms or equivalents, unless it is explicitlystated. Use of examples, including examples of terms, is forillustrative purposes only and does not limit the scope and meaning ofthe embodiments of the invention herein.

Briefly, and as described in more detail below, described herein arecompositions of vault complexes containing cytokines, such as CCL-21,and their use in delivering the cytokines to a cell. The vault particlesinclude a fusion protein of the cytokine of interest fused to majorvault interaction domain. Also included in the invention is the use ofthe compositions as cancer immunotherapy agents for activating an immuneresponse against a tumor and for treating cancers, including lungcancer.

CCL-21 and other cytokines have been shown to be effective as cancerimmunotherapy agents. However, conventional approaches for cancertherapy treatment with cytokines, such as CCL-21, involve use ofmodified dendritic cells. These preparations and treatments arecumbersome, expensive and time consuming. Difficulties with conventionalmethods include autologous DC preparation, batch to batch variabilityand lack of comparability and standardization. More convenient andefficacious options for delivery and treatment with cytokine reagentsare required.

The invention supplies the deficiencies of the conventional DC-basedmethods. Vault complexes provide effective and efficient intratumoralcytokine, e.g., CCL-21 delivery for the purpose of initiating antitumorimmune responses.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “cytokine” is a protein that is a member of a family ofsecreted cell-signaling proteins involved in immunoregulatory andinflammatory processes. A “chemokine” is a member of a family ofcytokines defined by invariant cysteine residues that form disulfidebonds. One example of a chemokine is “CCL-21” referring to a chemokine(C-C motif) ligand 21. A C-C motif is a cysteine-cysteine motif.

As used herein, the term “vault” or “vault particle” refers to a largecytoplasmic ribonucleoprotein (RNP) particle found in eukaryotic cells.The vault or vault particle is composed of MVP, VPARP, and/or TEP1proteins and one or more untranslated vRNA molecules.

As used herein, the term “vault complex” refers to a recombinant vaultthat encapsulates a small molecule or protein of interest. A vaultcomplex of the invention includes a fusion protein, e.g., a cytokinefusion protein.

As used herein, the term “cytokine fusion protein” is a recombinantprotein expressed from a nucleotide encoding a cytokine fused in frameto a vault targeting domain.

As used herein, the term “vault targeting domain” or “vault interactiondomain” is a domain that is responsible for interaction or binding of aheterologous fusion protein with a vault protein, or interaction of aVPARP with a vault protein, such as a MVP. As used herein, the term“mINT domain” is a vault interaction domain from a vault poly ADP-ribosepolymerase (VPARP) that is responsible for the interaction of VPARP witha major vault protein (MVP). The term “mINT domain” refers to a majorvault protein (MVP) interaction domain.

As used herein, the term “MVP” is major vault protein. The term “cp-MVP”is a cysteine-rich peptide major vault protein.

The term “VPARP” refers to a vault poly ADP-ribose polymerase.

As used herein, the term “TEP-1” is a telomerase/vault associatedprotein 1.

As used herein, the term “vRNA” is an untranslated RNA molecule found invaults.

As used herein, the term “fluorescent protein” is a protein that has theproperty of forming a visible wavelength chromophore from within itspolypeptide sequence. Fluorescent proteins can be engineered to beexpressed with other proteins, and include, but are not limited to,green fluorescent protein (GFP), red fluorescent protein (mCherry), bluefluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescentprotein (ECFP, Cerulean, CyPet) and yellow fluorescent proteinderivatives (YFP, Citrine, Venus, YPet).

As used herein, the term “vector” is a DNA or RNA molecule used as avehicle to transfer foreign genetic material into a cell. The four majortypes of vectors are plasmids, bacteriophages and other viruses,cosmids, and artificial chromosomes. Vectors can include an origin ofreplication, a multi-cloning site, and a selectable marker.

As used herein, a “cell” includes eukaryotic and prokaryotic cells.

As used herein, the terms “organism”, “tissue” and “cell” includenaturally occurring organisms, tissues and cells, genetically modifiedorganisms, tissues and cells, and pathological tissues and cells, suchas tumor cell lines in vitro and tumors in vivo.

As used herein, the term “T cell” or T lymphocyte is a white blood cellknown as a lymphocyte, and plays a central role in cell-mediatedimmunity.

As used herein, the term “extracellular environment” is the environmentexternal to the cell.

As used herein, the term “in vivo” refers to processes that occur in aliving organism.

A “subject” referred to herein can be any animal, including a mammal(e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit,primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat,donkey, sheep, etc.), a domestic animal (e.g., cat, dog, ferret, etc.),an avian species, or a human.

The term “mammal” as used herein includes both humans and non-humans andinclude but is not limited to humans, non-human primates, canines,felines, murines, bovines, equines, and porcines.

As used herein, the term “human” refers to “Homo sapiens.”

As used herein, the term “sufficient amount” is an amount sufficient toproduce a desired effect, e.g., an amount sufficient to modulate proteinaggregation in a cell.

As used herein, the term “therapeutically effective amount” is an amountthat is effective to ameliorate a symptom of a disease, such as cancer.

A “prophylactically effective amount” refers to an amount that iseffective for prophylaxis.

An “immune response” is a response by a host against foreign immunogensor antigens. A “cell-mediated immune response” refers to a helper T cellresponse which involves the production of interferon-gamma (IFN-γ),leading to cell-mediated immunity.

As used herein, the term “stimulating” refers to activating, increasing,or triggering a molecular, cellular or enzymatic activity or responsefrom within a cell or organism.

As used herein, the term “administering” includes any suitable route ofadministration, as will be appreciated by one of ordinary skill in theart with reference to this disclosure, including direct injection into asolid organ, direct injection into a cell mass such as a tumor,inhalation, intraperitoneal injection, intravenous injection, topicalapplication on a mucous membrane, or application to or dispersion withinan environmental medium, and a combination of the preceding.

As used in this disclosure, the term “modified” and variations of theterm, such as “modification,” means one or more than one change to thenaturally occurring sequence of MVP, VPARP or TEP1 selected from thegroup consisting of addition of a polypeptide sequence to theC-terminal, addition of a polypeptide sequence to the N-terminal,deletion of between about 1 and 100 amino acid residues from theC-terminal, deletion of between about 1 and 100 amino acid residues fromthe N-terminal, substitution of one or more than one amino acid residuethat does not change the function of the polypeptide, as will beappreciated by one of ordinary skill in the art with reference to thisdisclosure, such as for example, an alanine to glycine substitution, anda combination of the preceding.

As used herein, the term percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refers to two or moresequences or subsequences that have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms described below (e.g., BLASTP and BLASTNor other algorithms available to persons of skill) or by visualinspection. Depending on the application, the percent “identity” canexist over a region of the sequence being compared, e.g., over afunctional domain, or, alternatively, exist over the full length of thetwo sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

As used in this disclosure, the term “comprise” and variations of theterm, such as “comprising” and “comprises,” are not intended to excludeother additives, components, integers or steps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Compositions of the Invention

As described in more detail below, the invention includes compositionsand methods of using vault particles. The vault particles arerecombinant particles having a MVP and a fusion protein and mINT and aprotein of interest, e.g., a cytokine, e.g., CCL-21. The vault particlecan be used for delivery of the protein of interest, e.g., the cytokine,to a cell or tumor or subject.

Vaults and Vault Complexes

The compositions of the invention comprise a vault complex. A vaultcomplex is a recombinant particle that encapsulates a small molecule(drug, sensor, toxin, etc.), or a protein of interest, e.g., a peptide,or a protein, including an endogenous protein, a heterologous protein, arecombinant protein, or recombinant fusion protein. Vault complexes areof the invention include a cytokine recombinant fusion protein. Vaultcomplexes are derived from vault particles.

Vaults, e.g., vault particles are ubiquitous, highly conservedribonucleoprotein particles found in nearly all eukaryotic tissues andcells, including dendritic cells (DCs), endometrium, and lung, and inphylogeny as diverse as mammals, avians, amphibians, the slime moldDictyostelium discoideum, and the protozoan Trypanosoma brucei(Izquierdo et al., Am. J. Pathol., 148(3):877-87 (1996)). Vaults have ahollow, barrel-like structure with two protruding end caps, aninvaginated waist, and regular small openings surround the vault cap.These openings are large enough to allow small molecules and ions toenter the interior of the vault. Vaults have a mass of about 12.9±1 MDa(Kedersha et al., J. Cell Biol., 112(2):225-35 (1991)) and overalldimensions of about 42×42×75 nm (Kong et al., Structure, 7(4):371-9(1999)). The volume of the internal vault cavity is approximately 50×10³nm³, which is large enough to enclose an entire ribosomal protein.

Vaults comprise three different proteins, designated MVP, VPARP andTEP1, and comprise one or more different untranslated RNA molecules,designated vRNAs. The number of vRNA can vary. For example, the ratRattus norvegicus has only one form of vRNA per vault, while humans havethree forms of vRNA per vault. The most abundant protein, major vaultprotein (MVP), is a 95.8 kDa protein in Rattus norvegicus and a 99.3 kDaprotein in humans which is present in 96 copies per vault and accountsfor about 75% of the total protein mass of the vault particle. The twoother proteins, the vault poly-ADP ribose polymerase, VPARP, a 193.3 kDaprotein in humans, and the telomerase/vault associated protein 1, TEP1,a 292 kDa protein in Rattus norvegicus and a 290 kDa protein in humans,are each present in between about 2 and 16 copies per vault.

VPARP, mINT Domain, and mINT Fusion Proteins

A vault poly ADP-ribose polymerase (VPARP) includes a region of about350 amino acids that shares 28% identity with the catalytic domain ofpoly ADP-ribosyl polymerase, PARP, a nuclear protein that catalyzes theformation of ADP-ribose polymers in response to DNA damage. VPARPcatalyzes an NAD-dependent poly ADP-ribosylation reaction, and purifiedvaults have poly ADP-ribosylation activity that targets MVP, as well asVPARP itself. VPARP includes a mINT domain (major vault protein (MVP)interaction domain). The mINT domain is responsible for the interactionof VPARP with a major vault protein (MVP).

A vault complex of the invention includes a mINT domain. The mINT domainis responsible for interaction of a protein of interest, e.g., acytokine, with a vault protein such as a MVP. In general, the mINTdomain is expressed as a fusion protein with a protein of interest,e.g., a cytokine. The mINT of the vault complexes of the invention arederived from VPARP sequences. Exemplary VPARP sequences and mINTsequences can be found in Table 1. One of skill in the art understandsthat the mINT can have the entire naturally occurring sequence orportions of the sequence or fragments thereof. In other embodiments, themINT has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity to any of the VPARP and/or mINT sequences disclosed inTable 1.

In one embodiment, the mINT is derived from a human VPARP, SEQ ID NO:14,GenBank accession number AAD47250, encoded by the cDNA, SEQ ID NO:15,GenBank accession number AF158255. In some embodiments, the vaulttargeting domain comprises or consists of the INT domain correspondingto residues 1473-1724 (SEQ ID NO:69) of human VPARP protein sequence(full human VPARP amino acid sequence is SEQ ID NO:14). In otherembodiments, the vault targeting domain comprises or consists of themINT domain comprising residues 1563-1724 (SEQ ID NO: 8) of the humanVPARP protein sequence. In certain embodiments, the vault targetingdomain comprises or consists of a mINT domain (SEQ ID NO: 6) (mousemINT). In some embodiments, the vault targeting domain comprises orconsists of SEQ ID NO: 7 (human mINT). In certain embodiments, the vaulttargeting domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NOs: 6 or 7.

In alternative embodiments, the mINT domain is derived from TEP1sequences. One of skill in the art understands that the mINT can havethe entire naturally occurring sequence of the vault interaction domainin TEP1 or portions of the sequence or fragments thereof.

MVP

A vault complex of the invention generally includes an MVP. ExemplaryMVP sequences can be found in Table 1. One of skill in the artunderstands that the MVP can have the entire naturally occurringsequence or portions of the sequence or fragments thereof. In otherembodiments, the MVP has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% sequence identity to any of the MVP sequences disclosedin Table 1.

In one embodiment, the MVP is human MVP, SEQ ID NO:16, GenBank accessionnumber CAA56256, encoded by the cDNA, SEQ ID NO:17, GenBank accessionnumber X79882. In another embodiment, the MVP is Rattus norvegicus MVP,SEQ ID NO:18, GenBank accession number AAC52161, encoded by the cDNA,SEQ ID NO:19, GenBank accession number U09870. In other embodiments, theMVP is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the MVP sequences described herein.

In one embodiment, there is provided a vault complex comprising,consisting essentially of, or consisting of an MVP modified by adding apeptide to the N-terminal to create a one or more than one of heavymetal binding domains. In a preferred embodiment, the heavy metalbinding domains bind a heavy metal selected from the group consisting ofcadmium, copper, gold and mercury. In a preferred embodiment, thepeptide added to the N-terminal is a cysteine-rich peptide (CP), such asfor example, SEQ ID NO:20, the MVP is human MVP, SEQ ID NO:16, and themodification results in CP-MVP, SEQ ID NO:21, encoded by the cDNA, SEQID NO:22. In another preferred embodiment, the cysteine-rich peptide isSEQ ID NO:20, the MVP is Rattus norvegicus MVP, SEQ ID NO:18, and themodification results in CP-MVP, SEQ ID NO:23, encoded by the cDNA, SEQID NO:24. These embodiments are particularly useful because vaultparticles consisting of CP-MVP, SEQ ID NO:21 or SEQ ID NO:23, are stablewithout the presence of other vault proteins.

Any of the vault complexes described herein can include MVPs or modifiedMVPs disclosed herein.

TEP1

In some embodiments, a vault particle of the invention includes a TEP1protein. Exemplary TEP1 sequences can be found in Table 1. One of skillin the art understands that the TEP1 can have the entire naturallyoccurring sequence or portions of the sequence or fragments thereof. Inother embodiments, the TEP1 has at least 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% sequence identity to any of the TEP1 sequencesdisclosed in Table 1.

The TEP1 can be human TEP1, SEQ ID NO:25, GenBank accession numberAAC51107, encoded by the cDNA, SEQ ID NO:26, GenBank accession numberU86136. In another embodiment, the TEP1 is Rattus norvegicus TEP1, SEQID NO:27, GenBank accession number AAB51690, encoded by the cDNA, SEQ IDNO:28, GenBank accession number U89282. Any of the vault complexesdescribed herein can include TEP1 or modifications thereof.

vRNA

A vault complex of the invention can include a vRNA. Exemplary vRNAsequences can be found in Table 1. One of skill in the art understandsthat the vRNA can have the entire naturally occurring sequence orportions of the sequence or fragments thereof. In other embodiments, thevRNA has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity to any of the vRNA sequences disclosed in Table 1.

In one embodiment, the vRNA can be a human vRNA, SEQ ID NO:29, GenBankaccession number AF045143, SEQ ID NO:30, GenBank accession numberAF045144, or SEQ ID NO:31, GenBank accession number AF045145, or acombination of the preceding. In another embodiment, the vRNA is Rattusnorvegicus vRNA, SEQ ID NO:32, GenBank accession number Z1171.

As will be appreciated by one of ordinary skill in the art withreference to this disclosure, the actual sequence of any of MVP, VPARP,TEP1 and vRNAs can be from any species suitable for the purposesdisclosed in this disclosure, even though reference or examples are madeto sequences from specific species. For example, when deliveringchemokines or cytokines to human organs or tissues, it is preferred touse human vaults or vault-like particles comprising human sequences forMVP, VPARP, TEP1 and vRNAs. Further, as will be appreciated by one ofordinary skill in the art with reference to this disclosure, there aresome intraspecies variations in the sequences of MVP, VPARP, TEP1 andvRNAs that are not relevant to the purposes of the present invention.Therefore, references to MVP, VPARP, TEP1 and vRNAs are intended toinclude such intraspecies variants.

Cytokines

The compositions of the invention include a vault complex including acytokine. In general, the vault complex includes a cytokine fusionprotein.

Cytokines are a family of secreted cell-signaling proteins involved inimmunoregulatory and inflammatory processes, which are secreted by theglial cells of the nervous system and by numerous cells of the immunesystem. Cytokines can be classified as proteins, peptides orglycoproteins, and encompass a large and diverse family of regulators.Cytokines bind to cell surface receptors to trigger intracellularsignaling, which can result in upregulation or downregulation of severalgenes and their transcription factors, or feedback inhibition.

In certain embodiments, the cytokines of the invention includeimmunomodulating agents, such as interleukins (IL) and interferons(IFN). Suitable cytokines can include proteins from one or more of thefollowing types: the four α-helix bundle family (which includes the IL-2subfamily, the IFN subfamily, and the IL-10 subfamily); the IL-1 family(which includes IL-1 and IL-8), and the IL-17 family. Cytokines can alsoinclude those classified as type 1 cytokines, which enhance cellularimmune responses (e.g., IFN-γ, TGF-β, etc.), or type 2 cytokines, whichfavor antibody responses (e.g., IL-4, IL-10, IL-13, etc.).

In one embodiment, the cytokine is a chemokine Chemokines are thelargest family of cytokines and are defined by four invariant cysteineresidues that form disulfide bonds. Chemokines function by activatingspecific G protein-coupled receptors, which results in the migration ofinflammatory and noninflammatory cells to the appropriate tissues orcompartments within tissues. The role of chemokines is to act as achemoattractant to guide the migration of cells and to promoteaccumulation of cells at the source of chemokine production.

In some embodiments, the cytokines of the invention include homeostaticchemokines, which are constitutively produced and secreted. Homeostaticchemokines direct trafficking of lymphocytes to lymphoid tissues and areinvolved in immune surveillance and function to localize T cells or Bcells with an antigen in the lymphatic system. In other embodiments, thechemokines of the invention include inflammatory chemokines that promoterecruitment and localization of dendritic cells to sites of inflammationand infection. Several chemokines are involved in migration of monocytesand immature dendritic cells, which express chemokine receptors such asCCR1, CCR2, CCR5, CCR6, CCR7 and CXCR2. Chemokine receptor expression isregulated on these dendritic cells. Upon exposure to maturation signals,dendritic cells undergo a chemokine receptor switch, with downregulationof inflammatory chemokine receptors followed by induction of CCR7. Thisallows immature dendritic cells to leave tissues and to localize inlymphoid organs (due to CCR7 agonists), where antigen presentation takesplace.

In certain embodiments, the cytokine comprises CC or β-chemokines, whichhave the first two cysteines adjacent to each other. In otherembodiments, the chemokine comprises CXC or α chemokines, which have anintervening amino acid between the first two cysteines. In otherembodiments, the chemokine comprises a CX3C or γ-chemokine, whichpossess only one protein in its category and is defined by threeintervening residues between the first two cysteines. One of twoexceptions to the four-cysteine paradigm is the C or 6-chemokine, inwhich the polypeptide has only two of the four cysteines.

In some embodiments, the cytokine comprises a CC chemokine. The CCchemokine is characterized by two adjacent cysteines near the aminoterminus and is also called a β-chemokine or 17q chemokine. The CCsubfamily includes at least 27 distinct members of the subfamily inmammals. These include, but are not limited to the following CCchemokines: CCL-1, CCL-2, CCL-3, CCL-4, CCL-5, CCL-7, CCL-8,CCL-9/CCL-10, CCL-11, CCL-12, CCL-13, CCL-14, CCL-15, CCL-16, CCL-17,CCL-18, CCL-19, CCL-20, CCL-21, CCL-22, CCL-23, CCL-24, CCL-25, CCL-26,CCL-27 and CCL-28. Chemokines of this subfamily usually contain fourcysteines (C4-CC chemokines), but a small number of CC chemokinespossess six cysteines (C6-CC chemokines). C6-CC chemokines include CCL1,CCL15, CCL21, CCL23 and CCL28. CC chemokines inhibit hemopoiesis andinduce the migration of monocytes and other cell types such as naturalkiller (NK) cells and dendritic cells. CC Chemokines are chemotactic invitro for thymocytes and activated T cells, but not for B cells,macrophages, or neutrophils. CC Chemokines may also play a role inmediating homing of lymphocytes to secondary lymphoid organs.

In other embodiments, the cytokine comprises a CXC chemokine CXCchemokines have two N-terminal cysteines separated by an amino acid “X”.There are 17 different CXC chemokines in mammals and are separated intwo categories, those with a specific amino acid sequence (or motif) ofglutamic acid-leucine-arginine (or ELR for short) immediately before thefirst cysteine of the CXC motif (ELR-positive), and those without an ELRmotif (ELR-negative). Other CXC chemokines that lack the ELR motif, suchas CXCL13, tend to be chemoattractant for lymphocytes. CXC chemokinesbind to CXC chemokine receptors, of which seven have been discovered todate, designated CXCR1-7.

In another embodiment, the cytokine comprises a C chemokine (also calledγ chemokine), which has only two cysteines (one N-terminal cysteine andone cysteine downstream). Two chemokines are included in this subgroup(XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β)). These chemokinesattract T cell precursors to the thymus.

In yet another embodiment, the cytokine comprises a CX₃C chemokine (ord-chemokines). The CX₃C chemokine has three amino acids between the twocysteines. The only CX₃C chemokine discovered to date is calledfractalkine (or CX₃CL1).

In some embodiments, the cytokine comprises a CCL-21 protein. CCL-21stands for chemokine (C-C motif) ligand 21 and is a member of the CCchemokine family. CCL-21 is encoded by the Scya21 gene and is alsocalled secondary lymphoid-tissue chemokine (SLC), 6Ckine, Exodus-2,Ckβ9, and TCA-4. The CCL-21 binds to the CCR7 receptor, a cell surfacechemokine receptor. The human CCL-21 gene is found on the p-arm ofchromosome 9 and has the Genbank Accession No. NP_(—)002980.

Exemplary cytokine sequences can be found in Table 1. One of skill inthe art understands that the cytokine can have the entire naturallyoccurring sequence or portions of the sequence or fragments thereof. Inother embodiments, the cytokine has at least 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98% or 99% sequence identity to any of the cytokinesequences disclosed in Table 1.

In some embodiments, the cytokine comprises or consists of SEQ ID NO: 1(mouse CCL-21 protein sequence). In other embodiments, the cytokinecomprises or consists of SEQ ID NO:2 (human CCL-21 protein sequence). Inother embodiments, the cytokine has at least 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs:1 or 2. Inanother embodiment, the cytokine of the invention is encoded by anucleic acid comprising SEQ ID NO:3 (mouse CCL-21 DNA sequence minus 3amino acids for stop codon) or SEQ ID NO:4 (full mouse CCL-21 DNAsequence). In yet another embodiment, the cytokine of the invention isencoded by a nucleic acid comprising SEQ ID NO:5 (human CCL-21 DNAsequence). In certain embodiments, the cytokine of the inventioncomprises the entire naturally occurring DNA sequence, portions of theDNA sequence or fragments thereof. In some embodiments, the cytokine ofthe invention is encoded by a nucleic acid comprising 50%, 60%, 70%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNOs:3, 4 or 5.

In yet another embodiment, the cytokine comprises one of any of thesequences of cytokines or chemokines in Table 1, shown below. Suitablecytokines from humans for use in compositions and methods of theinvention include, but are not limited to, interleukin-2 (IL-2) (DNAsequence is SEQ ID NO:33 and protein sequence is SEQ ID NO:34),interleukin-7 (IL-7) (DNA sequence is SEQ ID NO:35 and protein sequenceis SEQ ID NO:36), interleukin 15 (IL-15) (DNA sequence is SEQ ID NO:37and protein sequence is SEQ ID NO:38), interleukin 12B (IL-12B) (DNAsequence is SEQ ID NO:39 and protein sequence is SEQ ID NO:40),interleukin 12A (IL-12A) (DNA sequence is SEQ ID NO:41 and proteinsequence is SEQ ID NO:42), colony stimulating factor 2 (DNA sequence isSEQ ID NO:43 and protein sequence is SEQ ID NO:44), chemokine (C-X-Cmotif) ligand 9 (CXCL9) (DNA sequence is SEQ ID NO:45 and proteinsequence is SEQ ID NO:46), chemokine (C-X-C motif) ligand 10 (CXCL10)(DNA sequence is SEQ ID NO:47 and protein sequence is SEQ ID NO:48),interferon alpha-d (IFN-alpha) (DNA sequence is SEQ ID NO:49 and proteinsequence is SEQ ID NO:50), interferon-gamma IEF SSP 5111 (DNA sequenceis SEQ ID NO:51 and protein sequence is SEQ ID NO:52), chemokine (C-Cmotif) ligand 19 (CCL-19) (DNA sequence is SEQ ID NO:53 and proteinsequence is SEQ ID NO:54), chemokine (C-C motif) ligand 21 (CCL-21) (DNAsequence is SEQ ID NO:55 and protein sequence is SEQ ID NO:56), tumornecrosis factor (TNF) (DNA sequence is SEQ ID NO:57 and protein sequenceis SEQ ID NO:58), and interleukin 27 (IL-27) (DNA sequence is SEQ IDNO:59 and protein sequence is SEQ ID NO:60).

As will be appreciated by one of ordinary skill in the art withreference to this disclosure, the actual sequence of any of cytokine canbe from any species suitable for the purposes disclosed in thisdisclosure, even though reference or examples are made to sequences fromspecific species. For example, when delivering chemokines or cytokinesto human organs or tissues, it is preferred to use human cytokines.Further, as will be appreciated by one of ordinary skill in the art withreference to this disclosure, there are some intraspecies variations inthe sequences of cytokine that are not relevant to the purposes of thepresent invention. Therefore, references to cytokine are intended toinclude such intraspecies variants.

Fusion Proteins

In general, the vault complexes of the invention include a fusionprotein, e.g., a cytokine fusion protein. The cytokine fusion protein isa recombinant protein expressed from a nucleotide encoding a chemokineor cytokine fused in frame to a vault targeting domain, e.g., mINT. Insome embodiments, the cytokine fusion protein comprises a mINT domainfused to a chemokine protein sequence. In other embodiments, thecytokine fusion protein comprises a mINT domain fused to a CCL-21protein. In another embodiment, the cytokine is fused to the N-terminusof an MVP protein. In one embodiment, the cytokine is fused to theC-terminus of the MVP protein.

Exemplary cytokine fusion sequences can be found in Table 1. One ofskill in the art understands that the cytokine fusion sequences can havethe entire naturally occurring sequence or portions of the sequence orfragments thereof. In other embodiments, the cytokine fusion sequencehas at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity to any of the cytokine fusion sequences disclosed in Table 1.

In certain embodiments, the cytokine fusion protein is encoded by thenucleic acid sequence of SEQ ID NO: 10 (mouse CCL21-mINT fusion DNAsequence). In other embodiments, the cytokine fusion protein is encodedby the nucleic acid sequence of SEQ ID NO: 11 (human CCL21-mINT fusionDNA sequence). In some embodiments, the cytokine fusion proteincomprises or consists of SEQ ID NO:12 (mouse CCL-21-mINT fusion proteinsequence). In some embodiments, the cytokine fusion protein comprises orconsists of SEQ ID NO: 13 (human CCL-21-mINT fusion protein sequence).

In one embodiment, the cytokine fusion protein includes the entirenaturally occurring cytokine protein sequence, a portion of the cytokineprotein sequence, or fragments thereof. In other embodiments, thecytokine fusion protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NOs: 12 or 13. In anotherembodiment, the cytokine fusion recombinant DNA sequence includes theentire naturally occurring cytokine DNA sequence, a portion of thecytokine DNA sequence, or fragments thereof.

Any of the cytokines described herein can be expressed as a fusionprotein with any of the mINT domain disclosed herein.

Fluorescent Proteins

In certain embodiments, the vault complex of the invention includes afluorescent protein. In some embodiments, the cytokine fusion proteincomprises a fluorescent protein. Fluorescent proteins can be engineeredto be expressed with other proteins, and include, but are not limitedto, green fluorescent protein (GFP), red fluorescent protein (mCherry),blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyanfluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescentprotein derivatives (YFP, Citrine, Venus, YPet). In one embodiment, thecytokine fusion protein comprises a mCherry fluorescent protein or aportion of a mCherry fluorescent protein.

Isolated Nucleic Acids and Vectors

The invention also includes isolated nucleic acid encoding a cytokinefusion protein comprising a cytokine encoding sequence and a vaulttargeting domain encoding sequence. In one embodiment, the isolatednucleic acid encodes a chemokine fusion protein comprising a CCL-21encoding sequence and a mINT encoding sequence. In another embodiment,the chemokine encoding sequence comprises or consists of SEQ ID NO:5(human) and the mINT encoding sequence consists of SEQ ID NO:7 (human).In another embodiment, the chemokine encoding sequence comprises orconsists of SEQ ID NO:3 (mouse) and the mINT encoding sequence consistsof SEQ ID NO:6 (mouse). In one embodiment, the isolated nucleic acid isa cDNA plasmid construct encoding the full length cytokine protein and amINT domain comprising or consisting of SEQ ID NO: 6 or 7 (human andmouse mINT). Table 1 lists nucleic acid sequences encoding someexemplary chemokine or cytokine fusion proteins.

The nucleic acid molecules encoding a cytokine fusion protein of theinvention can be expressed from a vector, such as a recombinant viralvector. The recombinant viral vectors of the invention comprisesequences encoding the cytokine fusion protein of the invention and anysuitable promoter for expressing the cytokine fusion sequences. Suitablepromoters include, for example, the U6 or H1 RNA pol III promotersequences and the cytomegalovirus promoter. Selection of other suitablepromoters is within the skill in the art. The recombinant viral vectorsof the invention can also comprise inducible or regulatable promotersfor expression of the cytokine fusion recombinant genes in a particulartissue or in a particular intracellular environment. In one embodiment,recombinant baculoviruses and promoters can be used from pFastBacplasmid and the Bac-to-Bac protocol (Invitrogen, Gaithersburg, Md., Cat.No. 13459-016 or 10608-016).

Suitable expression vectors generally include DNA plasmids or viralvectors. Expression vectors compatible with eukaryotic cells, preferablythose compatible with vertebrate cells, can be used to producerecombinant constructs for the expression of an iRNA as describedherein. Eukaryotic cell expression vectors are well known in the art andare available from a number of commercial sources. Typically, suchvectors are provided containing convenient restriction sites forinsertion of the desired nucleic acid segment. Delivery of expressionvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

Plasmids expressing a nucleic acid sequence encoding a cytokine fusionprotein can be transfected into target cells as a complex with cationiclipid carriers (e.g., Oligofectamine) or non-cationic lipid-basedcarriers (e.g., Transit-TKO™). Successful introduction of vectors intohost cells can be monitored using various known methods. For example,transient transfection can be signaled with a reporter, such as afluorescent marker, such as Green Fluorescent Protein (GFP). Stabletransfection of cells ex vivo can be ensured using markers that providethe transfected cell with resistance to specific environmental factors(e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct maybe incorporated into vectors capable of episomal replication, e.g., EPVand EBV vectors. Constructs for the recombinant expression of a nucleicacid encoding a cytokine fusion protein will generally requireregulatory elements, e.g., promoters, enhancers, etc., to ensure theexpression of the cytokine fusion nucleic acid in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of a cytokine fusion nucleic acid caninclude regulatory elements (promoter, enhancer, etc.) sufficient forexpression of the cytokine fusion nucleic acid in the desired targetcell or tissue. The regulatory elements can be chosen to provide eitherconstitutive or regulated/inducible expression. A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the transgene.

In a specific embodiment, viral vectors that contain the recombinantgene can be used. For example, a retroviral vector can be used (seeMiller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviralvectors contain the components necessary for the correct packaging ofthe viral genome and integration into the host cell DNA. The nucleicacid sequences encoding a cytokine fusion protein are cloned into one ormore vectors, which facilitates delivery of the nucleic acid into apatient. More detail about retroviral vectors can be found, for example,in Boesen et al., Biotherapy 6:291-302 (1994), which describes the useof a retroviral vector to deliver the mdr1 gene to hematopoietic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993). Lentiviral vectors contemplated for useinclude, for example, the HIV based vectors described in U.S. Pat. Nos.6,143,520; 5,665,557; and 5,981,276, which are herein incorporated byreference.

Adenoviruses are also contemplated for use in delivery of isolatednucleic acids encoding cytokine fusion proteins into a cell.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia or for use in adenovirus-based delivery systemssuch as delivery to the liver, the central nervous system, endothelialcells, and muscle. Adenoviruses have the advantage of being capable ofinfecting non-dividing cells. Kozarsky and Wilson, Current Opinion inGenetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing a nucleic acid molecule featured in theinvention, a method for constructing the recombinant AV vector, and amethod for delivering the vector into target cells, are described in XiaH et al. (2002), Nat. Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walshet al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No.5,436,146). Suitable AAV vectors for expressing the dsRNA featured inthe invention, methods for constructing the recombinant AV vector, andmethods for delivering the vectors into target cells are described inSamulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al.(1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941;International Patent Application No. WO 94/13788; and InternationalPatent Application No. WO 93/24641, the entire disclosures of which areherein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus,for example an attenuated vaccinia such as Modified Virus Ankara (MVA)or NYVAC, an avipox such as fowl pox or canary pox.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

Examples of additional expression vectors that can be used in theinvention include pFASTBAC expression vectors and E. coli pET28aexpression vectors.

Generally, recombinant vectors capable of expressing genes forrecombinant cytokine fusion proteins are delivered into and persist intarget cells. The vectors or plasmids can be transfected into targetcells by a transfection agent, such as Lipofectamine. Examples of cellsuseful for expressing the nucleic acids encoding the cytokine fusionproteins of the invention include Sf9 cells or insect larvae cells.Recombinant vaults based on expression of the MVP protein alone can beproduced in insect cells. Stephen, A. G. et al. (2001). J. Biol. Chem.276:23217:23220; Poderycki, M. J., et al. (2006). Biochemistry (Mosc).45: 12184-12193.

Pharmaceutical Compositions of the Invention

In one embodiment, the invention provides methods using pharmaceuticalcompositions comprising the vault complexes of the invention. Thesecompositions can comprise, in addition to one or more of the vaultcomplexes, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material can depend on the route of administration, e.g. oral,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraperitoneal routes.

In certain embodiments, the pharmaceutical compositions that areinjected intra-tumorally comprise an isotonic or other suitable carrierfluid or solution.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives can be included, as required.

In other embodiments, pharmaceutical compositions for oraladministration can be in tablet, capsule, powder or liquid form. Atablet can include a solid carrier such as gelatin or an adjuvant.Liquid pharmaceutical compositions generally include a liquid carriersuch as water, petroleum, animal or vegetable oils, mineral oil orsynthetic oil. Physiological saline solution, dextrose or othersaccharide solution or glycols such as ethylene glycol, propylene glycolor polyethylene glycol can be included.

In some embodiments, administration of the pharmaceutical compositionsmay be topical, pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal, oral or parenteral. Parenteral administrationincludes intravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g.,intraparenchymal, intrathecal or intraventricular, administration.Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. For intravenous use, the total concentration of solutesshould be controlled to render the preparation isotonic.

Methods of Use

Vault complexes described herein can be used to deliver a protein ofinterest, e.g., cytokines, to a cell, a tissue, an environment outside acell, a tumor, an organism or a subject. In one embodiment, the vaultcomplex comprises a cytokine described herein, e.g., CCL-21, and thevault complex is introduced to the cell, tissue, or tumor. In someembodiments, the vault complex is introduced into the extracellularenvironment surrounding the cell. In other embodiments, the vaultcomplex is introduced into an organism or subject. Delivery of the vaultcomplex of the invention can include administering the vault complex toa specific tissue, specific cells, an environmental medium, or to theorganism. In some embodiments, delivery of the vault complex can bedetected by a sensor within the cell, tissue, or organism. For example,detection can be performed using standard techniques, such asfluorometry or spectrophotometry. This method can be used, for example,to determine the pH within cells, where the sensor is a pH dependentfluorescent sensor, as will be appreciated by one of ordinary skill inthe art with reference to this disclosure.

The methods of the invention comprise stimulating an immune response toa cell by contacting the cell with any of the vault complexes describedherein. Cells of the invention can include, but are not limited to, anyeukaryotic cell, mammalian cell, or human cells, including tumor cells.In some embodiments, contacting the cell with a vault complex inducesmigration of T cells and/or dendritic cells to the cell.

Methods of the invention include delivery of the vault complex to asubject. The delivery of a vault complex to a subject in need thereofcan be achieved in a number of different ways. In vivo delivery can beperformed directly by administering a vault complex to a subject.Alternatively, delivery can be performed indirectly by administering oneor more vectors that encode and direct the expression of the vaultcomplex or components of the vault complex. In one embodiment, the vaultcomplex is administered to a mammal, such as a mouse or rat. In anotherembodiment, the vault complex is administered to a human.

In one embodiment, the methods of delivery of the invention includesystemic injection of vault complexes to tumors, producing the enhancedpermeability and retention (EPR) effect. See Maeda et al., J. ofControlled Release 2000, 65: 271-284; Griesh, K., J. of Drug Targeting2007, 15(7-8): 457-464; Allen et al., Science 2004, 303:1818-1822. Solidtumors possess extensive angiogenesis and hence hypervasculature,defective vascular architecture, impaired lymphatic drainage/recoverysystems, and greatly increased production of a number of permeabilitymediators. Due to the biology of solid tumors, macromolecular anticancerdrugs and agents, including vault complexes, administered intravenouslycan accumulate and are retained in the tumor due to the lack ofefficient lymphatic drainage in the solid tumor. The invention includesmethods of systemic or targeted delivery of vault complexes describedherein to solid tumors, such as those found in lung cancer.

Other methods of the invention include stimulating an immune response ina subject. The method comprises administering the vault complex to asubject. Administering can include intra-tumoral injection of the vaultcomplex in a subject, which is described in detail herein.

Methods of Treatment

The invention features a method of treating or managing disease, such ascancer, by administering the vault complex of the invention to a subject(e.g., patient). In some embodiments, the vault complexes of theinvention can be used for treating or managing lung cancer. In anotherembodiment, the method of the invention comprises treating or managingcancer in a subject in need of such treatment or management, comprisingadministering to the subject a therapeutically effective amount of thevault complexes described herein. In one embodiment, the method involvestreating a human by identifying a human diagnosed as having lung canceror at risk for developing lung cancer and administering to the human atherapeutically or prophylactically effective amount of the CCL-21 vaultcomplex to the human. In another embodiment, the method comprisesadministering to the human to therapeutically or prophylacticallyeffective amount of the CCL-21 vault complex by intra-tumoral injection.

Vault complexes of the invention can be used to treat any solid cancer,e.g., lung cancer, breast cancer, head and neck cancer, prostate cancer,etc. Advances in mouse genetics have generated a number of mouse modelsfor the study of various human diseases, such as treatment of lungcancer. Such models are used for in vivo testing of vault complexes, aswell as for determining a therapeutically effective dose. A suitablemouse model is, for example, a tumor-bearing mouse that is administeredan intra-tumoral injection of a CCL-21 vault complex.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the vault complex. Suchinformation can be used to more accurately determine useful doses inhumans. Analysis of tumor cell samples of mice administered a vaultcomplex can also indicate a therapeutically effective dose.

The pharmaceutical composition according to the present invention to begiven to a subject, administration is preferably in a “therapeuticallyeffective amount” or “prophylactically effective amount” (as the casecan be, although prophylaxis can be considered therapy), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of protein aggregation disease being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980. Acomposition can be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

In certain embodiments, the dosage of vault complexes is between about0.1 and 10,000 micrograms per kilogram of body weight or environmentalmedium. In another embodiment, the dosage of vault complexes is betweenabout 1 and 1,000 micrograms per kilogram of body weight orenvironmental medium. In another embodiment, the dosage of vaultcomplexes is between about 10 and 1,000 micrograms per kilogram of bodyweight or environmental medium. For intravenous injection andintraperitoneal injection, the dosage is preferably administered in afinal volume of between about 0.1 and 10 ml. For inhalation the dosageis preferably administered in a final volume of between about 0.01 and 1ml. As will be appreciated by one of ordinary skill in the art withreference to this disclosure, the dose can be repeated a one or multipletimes as needed using the same parameters to effect the purposesdisclosed in this disclosure.

For instance, the pharmaceutical composition may be administered oncefor each tumor in a subject, or the vault complex may be administered astwo, three, or more sub-doses or injections at appropriate intervals. Inthat case, the vault complexes can be injected in sub-doses in order toachieve the total required dosage.

The vault complexes featured in the invention can be administered incombination with other known agents effective in treatment of cancers,including lung cancer. An administering physician can adjust the amountand timing of vault complex administration or injection on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein. The skilled artisan will also appreciate that certainfactors may influence the dosage and timing required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present.

Methods of Preparing Vault Complexes

The methods of the invention include preparing the vault complexesdescribed herein.

In one embodiment, the vault complexes are derived or purified fromnatural sources, such as mammalian liver or spleen tissue, using methodsknown to those with skill in the art, such as for example tissuehomogenization, differential centrifugation, discontinuous sucrosegradient fractionation and cesium chloride gradient fractionation. Inanother embodiment, the vault complexes are made using recombinanttechnology. Details about the methods for recombinant vault complexesare described below.

In some embodiments, a target of interest, i.e., protein of interest, isselected for packaging in the vault complexes. The target of interestmay be selected from the group consisting of an enzyme, a pharmaceuticalagent, a plasmid, a polynucleotide, a polypeptide, a sensor and acombination of the preceding. In a preferred embodiment, the target ofinterest is a recombinant protein, e.g., a cytokine fusion protein,e.g., a CCL-21 fusion protein.

Preferably, if the target of interest is a recombinant protein, thepolynucleotide sequences encoding the recombinant protein are used togenerate a bacmid DNA, which is used to generate a baculoviruscomprising the sequence. The baculovirus is then used to infect insectcells for protein production using an in situ assembly system, such asthe baculovirus protein expression system, according to standardtechniques, as will be appreciated by one of ordinary skill in the artwith reference to this disclosure. Advantageously, the baculovirusprotein expression system can be used to produce milligram quantities ofvault complexes, and this system can be scaled up to allow production ofgram quantities of vault complexes according to the present invention.

In another embodiment, the target of interest is incorporated into theprovided vaults. In a preferred embodiment, incorporation isaccomplished by incubating the vaults with the target of interest at anappropriate temperature and for an appropriate time, as will beappreciated by one of ordinary skill in the art with reference to thisdisclosure. The vaults containing the protein of interest are thenpurified, such as, for example sucrose gradient fractionation, as willbe appreciated by one of ordinary skill in the art with reference tothis disclosure.

In other embodiments, the vaults comprising the target of interest areadministered to an organism, to a specific tissue, to specific cells, orto an environmental medium. Administration is accomplished using anysuitable route, as will be appreciated by one of ordinary skill in theart with reference to this disclosure.

In one embodiment, the method comprises preparing the composition of theinvention by a) mixing a fusion protein comprising a chemokine fused toa mINT generated in Sf9 cells with a rat MVP generated in Sf9 cells togenerate a mixture; b) incubating the mixture for a sufficient period oftime to allow packaging of the fusion protein inside of vault complexes,thereby generating the composition. Sf9 cells are infected withCCL-21-mCherry-mINT or CP-MVP encoding recombinant baculoviruses.Lysates containing recombinant CCL-21-mINT and rat MVP generated in Sf-9cells can be mixed to allow the formation of a macromolecular vaultcomplex containing the CCL-21 fusion protein.

In another embodiment, the composition is prepared by a) mixing a fusionprotein comprising a chemokine fused to a mINT generated in insectlarvae cells with a rat MVP generated in insect larvae cells to generatea mixture; b) incubating the mixture for a sufficient period of time toallow packaging of the fusion protein inside of vault complexes.

Details about methods of preparing vault complexes are further describedin the Examples.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Methods

Cloning, Expression, and Purification of Vault Complexes

A cDNA encoding CCL-21 was fused in frame to either mINT or mCherry-mINT[21]. Murine CCL-21 was PCR amplified using the following primers:CCL21-forward GCGCGGATCCCCATGGCTCAGATGATG (SEQ ID NO:63) andCCL-21-reverse GCGCAGATCTTCCTCTTGAGGGCTGTGTCTG (SEQ ID NO:64). To formmCCL-21-mCherry-mINT in pFastBac, the CCL21 PCR product was purified ona Qiagen column, digested with BamH1 and Bg1 I, gel purified, andligated to BamH1 phosphatase treated mCherry-mINT pFastBac. Human CCL21was PCR amplified with the following primers: CCL-21 F-SpeICCCCACTAGTCCAGTTCTCAGTCACTGGCTCTG (SEQ ID NO: 65), CCL-21-NheICCCCGCTAGCTGGCCCTTTAGGGGTCTGTG (SEQ ID NO:66), mINT with NheICCCCGCTAGCTGCACACAACACTGGCAGGA (SEQ ID NO:67), mINT with XhoIGGGGCTCGAGTTAGCCTTGACTGTAATGGA (SEQ ID NO:68) to form hCCL-21-mINT.

Recombinant baculoviruses were generated using the Bac-to-Bac protocol(Invitrogen, Gaithersburg, Md.). Sf9 cells were infected withCCL-21-mCherry-mINT or CP-MVP encoding recombinant baculoviruses at amultiplicity of infection (MOI) of 0.01 for 65 h. The infected cellswere pelleted, and lysed on ice in buffer A [50 mM Tris-HCl (pH 7.4), 75mM NaCl, and 0.5 mM MgCl₂] with 1% Triton X-100, 1 mM dithiotreitol, 0.5mM PMSF, and protease inhibitor cocktail (Sigma P8849). Lysatescontaining CP-MVP vaults were mixed with lysates containingmCCL-21-mCherry-mINT, hCCL-21-mINT and were incubated on ice for 30 minto allow the INT fusion proteins to package inside of vaults.Recombinant vault complexes were purified as previously described [7].Purified recombinant vault complexes were resuspended in 100-200 μl ofsterile phosphate buffered saline. Protein concentration was determinedusing the BCA assay (Bio-Rad Laboratories, Hercules, Calif.) and sampleintegrity was analyzed by negative stain electron microscopy andSDS-PAGE followed by Coomassie staining and Western blot analysis.

Antibodies

Primary antibodies for Western blot analyses were rabbit anti-MVPpolyclonal antibody (1/1000 dilution) or rabbit anti-VPARP polyclonalantibody (1/500 dilution, overnight) and secondary goat anti-rabbitHRP-conjugated antibodies (1:2000 dilution) (Amersham). The anti-CCL-21antibodies were purchased from R&D Systems, (Minneapolis, Minn.).Primary antibody for immunostaining for CD3 was purchased from DAKO.Fluorescein isothiocyanate-, phycoerythrin-, allophycocyanin-, PerCP- orPerCP-Cy7-conjugated anti-mouse mAbs to CD3 (145-2C11), CD4 (RM4-5),CD8a (53-6.7) and subclass control antibody, were purchased from BDBiosciences (San Diego, Calif.). Anti-mouse mAbs to detect Tregs withcell surface CD4 (GK1.5), CD25 (PC61), intranuclear Foxp3 (FJK-16s)IL-10 (JES5-16E3), and IFNγ (XMG1.2) were purchased from eBioScience(San Diego, Calif.) were purchased from eBioScience (San Diego, Calif.).Antibodies to DEC205 (205yekta), CCR7 (4B12) and EpCam (G8.8) were fromeBioScience. Antibody to mouse CD11b (M1/70), Gr1 (RB6-8C5), werepurchased from BioLegend (San Diego). Anti-mouse mAb to CXCR3 (220803),was purchased from R&D Systems (Minneapolis, Minn.). Ovalbumin proteinand Bradford protein quantification dye was obtained from Sigma (St.Louis, Mo.). Tissue digestion buffer consisted of [0.2 mg/ml ofCollagenase A (Boehringer Mannheim/Roche, Indianapolis, Ind.), DNase 25U/ml (Sigma), and 0.3 U/ml of Dispase (Invitrogen, Carlsbad, Calif.)] inRPMI.

Chemotaxis Assay

Dual-chamber chemotaxis assays were performed using 24-well plates with3 μm pore size inserts (Costar/Corning, Corning, N.Y., United States)according to the manufacturer's instructions. Briefly, 2.0×10⁵ T2 cellswere resuspended in serum-free medium and loaded in the upper chamber.200 ng/ml of CCL-21-mcherry-vault, 600 ng/ml recombinant CCL-21 (R&DSystems), 200 ng/ml CCL-21-mcherry-vault with neutralizing anti-CCL-21recombinant antibody (5 μg/ml), 600 ng/ml CCL-21 with neutralizinganti-CCL-21 antibody (5 μg/ml) were added to the lower chamber of thewells (in triplicate). The neutralizing concentration of anti-CCL-21antibody (R&D) used in these studies (5 μg/ml) was based on the ND50(50% maximum inhibition of cytokine activity when CCL-21 is present at aconcentration high enough to elicit maximum response). After 2 hoursincubation at 37° C., migrated cells were recovered from the lowerchamber and the inserts according to the manufacturer's instructions.Migrated T2 cells were resuspended in FACS buffer and evaluated bycounting the number of lymphocytes.

Antigen Processing and Presentation Assay

Cells (DC2.4 (5×10⁴ c/well)) were plated in triplicates in 96-wellplates with OVA protein (350 μg/ml), MHC Class I restricted CD8 T cellline B3Z (10⁵ c/well), in the presence of control vaults (200 ng/ml), orCCL-21 vault complex (200 ng/ml) or rCCL-21 (200 ng/ml) for 24 hrs. Todetermine the impact of CCL-21 on APC activity, CCL-21 was neutralizedwith anti-CCL-21 Ab (5 μg/ml) (R&D). IL-2 secreted by the activated CD8T cells in the supernatant was quantified by ELISA (eBioScience).

Cell Culture

The murine Lewis lung carcinoma cell line (3LL, H2^(b)) was obtainedfrom American Type Culture Collection (ATCC, Manassas, Va.). The cellswere routinely cultured as monolayers in 25-cm² tissue culture flaskscontaining RPMI 1640 medium (Irvine Scientific, Santa Ana, Calif.)supplemented with 10% FBS (Gemini Bioproducts, Calabasas, Calif.),penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 2 mM glutamine (JRHBiosciences, Lenexa, Kans.), and maintained at 37° C. in humidifiedatmosphere containing 5% CO₂ in air. The cell line was mycoplasma free,and cells were utilized before the tenth passage.

Tumorigenesis Model

Pathogen-free C57BL/6 mice and UBC-GFP/BL6 (6-8 wk old; JacksonLaboratory) were maintained in the West Los Angeles Veterans AffairsAnimal Research vivarium. For tumorigenesis experiments, 1.5×10⁵ 3LLtumor cells were injected s.c. in the right suprascapular area ofC57BL/6 mice. Mice bearing 9-day-old established tumors were treatedwith a single intratumoral injection of mCCL-21-mCherry-CP-MVP vaults(200 ng), CP-MVP vaults (200 ng) in 200 μl or normal saline diluents.Tumor volumes were monitored by measuring two bisecting diameters ofeach tumor with calipers. Tumor volumes were calculated using theformula: V=0.4 ab², with “a” as the larger diameter and “b” as thesmaller diameter. To determine the extent of lymphocytes infiltratingthe tumors, UBC-GFP/BL6 mice bearing 9-day tumors were treated asdescribed and 7 days post treatment, non-necrotic tumors were isolatedand frozen in OCT. The frozen tissue was sectioned to 5-μm thickness,fixed onto slides, and counterstained with 4′,6-diamidino-2-phenylindole(DAPI) fixative. The slides were observed under a 1X71 Olympusfluorescence microscope attached to a charge-coupled device camera. Theimages were acquired under x10 and x40 objectives using the Image Prosoftware.

Orthotopic Model

Implantation of the tumors in the lung was performed as previouslydescribed in Andersson, A. et al. J Immunol 2009, 182(11):6951-6958[24]. Briefly, 5×10³ 3LL-GFP cells in 25 μl NS diluent were injected bythe transthoracic route of C57BL/6 mice utilizing a tuberculin syringewith a 30-gauge needle in the left lung under ketamine/xylazineanesthesia. One week following tumor inoculation, mice were treated withdiluent, control vault or CCL-21 vault complex via transthoracicinjection. Four weeks after tumor implantation, lungs were harvested forevaluation of tumor burden and leukocytic infiltrates. Tumor burden wasquantified by gating on the GFP and EpCam expressing 3LL tumor cells insingle cell suspension of lung-tumor digests.

Immunostaining

Immunohistochemical staining was performed to determine and characterizethe infiltrating cells. Specifically, paraffin sections of 5 μm weredeparaffinized in xylene and rehydrated in decreasing concentrations ofethanol according to standard protocol [25]. Heat-induced antigenretrieval in citrate buffer (3 min in a steamer) was followed byblockade of endogenous peroxidase activity with 3% hydrogen peroxide inTBS for 10 min. All tissue was blocked (4% BSA, 10% sucrose, 1% normalswine serum in TBS) for 20 min at room temperature (RT). Primaryantibody (DAKO, Cytomation, Carpinteria, Calif., USA) was diluted in theblocking solution to the following concentrations: CD3 1:200. Sectionswere incubated with the antibodies overnight at 4° C. On the second day,the slides were washed with Tris-buffered saline containing 0.02% Tween.This was followed by incubation with secondary biotinylated goatanti-mouse antibody at room temperature, streptavidin-conjugatedalkaline phosphatase (Vectastain ABC-AP kit; Vector Laboratories,Burlingame, Calif.), and chromagen development with Vector Red substratesolution (Vector Laboratories). Slides were counterstained withhematoxylin, dehydrated, and mounted for analysis and photography.

Flow Cytometry

Flow cytometry was performed for the following leukocytic markers CD3,CD4, CD8, CCR7, CD11b, Gr1, DEC205, CD25, FOXP3 and CXCR3 on single cellsuspension of tumors following treatment as described above. T cellswere stained for intracytoplasmic IFNγ and IL-10. For analyses in thetumor tissue, tumors were mechanically dissociated on a wire mesh bycrushing with a 10 ml syringe and incubated in tissue digestion bufferat 37° C. for 25 min. The cells were filtered through 70 μm nylonstrainers (BD Biosciences, Bedford, Mass.) and stained with specificmarkers and analyzed by flow cytometry. Samples were acquired on aFACSCanto (BD Biosciences/FACSCalibur flow cytometer (Becton Dickinson,San Jose, Calif.) in the University of California, Los Angeles, JonssonCancer Center Flow Cytometry Core Facility. A total of 10,000 to 25,000gated events were analyzed using FCS Express 3 (De Novo Software,Canada). Cells incubated with irrelevant isotype-matched antibodies andunstained cells served as controls. The cutoffs were set according tocontrol staining.

T Cell Cytolysis

T lymphocyte lytic responses were evaluated following therapy. T cellswere purified from spleens by negative selection using Miltenyi Biotecbeads, and cytolytic activities were evaluated against autologous 3LLtumor cell line and the syngeneic control B16 melanoma tumor cell line.The T cell effectors were co-cultured with tumor cell targets (E:T of20:1 and 40:1) in quadruplet wells in a 96-well plate, and 20 μl alamarblue was added to each well after 18 hours of incubation. Three hoursafter alamar blue addition, the plate was read with the Wallac 1420fluorescence plate reader (Perkin-Elmer Life Science, Turku, Finland)with the excitation/emission set at 530/590 nm.

Example 1 Packaging CCL-21 into the Recombinant Vaults

A mouse chemokine CCL-21 was fused to a mouse mINT to create a CCL-21fusion protein that was packaged into vault complexes. FIG. 1A shows adiagram of the CCL-21 and mCherry-INT constructs (SEQ ID NO:60) thatwere fused to create the mouse CCL-21-mCherry-mINT fusion protein (SEQID NO: 61). Mixing of lysates containing recombinant CCL-21-mINT and ratMVP generated in Sf-9 cells allowed the formation of a macromolecularvault complex containing the CCL-21 fusion protein that could beisolated by density gradient ultracentrifugation. The purified vaultcomplexes contained both MVP as well as CCL-21-mINT (henceforth referredto as CCL-21 vault complex). FIG. 1B shows the incorporation of therecombinant CCL-21-mINT into a pFastBac expression vector by restrictiondigest and expression of CCL-21 fusion protein as analyzed on anelectrophoresis gel. The MVP recombinant vaults containing packagedCCL-21-mCherry-mINT were purified on a sucrose gradient and the 40 and45% fractions were analyzed by SDS-PAGE (FIG. 1C) and followed bystaining with Coomassie (FIG. 1D). FIG. 1E shows a negative stain TEMimage of vaults containing CCL-21-mCherry-mINT.

There was an estimated 20-30 molecules of the CCL-21-mINT protein ineach vault complex is based on extrapolation from densitometric analysisof the Coomassie stained SDS-PAGE gels. This is consistent with previousstudies in packaging multiple copies of other mINT fusion proteins intorecombinant vault complexes [21]. With an estimate of 20-30 CCL-21-mINTproteins per vault, it is likely that this is at or near a saturatinglevel for the packaging of this size protein. The CCL-21 vault complexalso exhibited a very similar sedimentation profile on sucrose gradientsas vault particles containing the INT domain fused to luciferase [6, 8,21], suggesting that incorporation of CCL-21-mINT did not impact thenormal structure of recombinant vault complexes. FIG. 1E shows purifiedvault complexes examined by negative stain transmission electronmicroscopy.

These results demonstrate that CCL-21 vaults complexes exhibit thecharacteristic barrel shaped morphology of vaults, consistent with thepreviously established morphology of vaults containing recombinant-INTfusion proteins [8, 26].

Example 2 CCL-21-Vault Complexes are Biologically Active and Induce theMigration of T2 Cells In Vitro

To determine whether CCL-21 retains its biological function whenpackaged inside the vault, a chemotaxis assay was used. The chemotacticactivity of CCL-21 is mediated through its receptor CCR7 to induce themigration of T cells and dendritic cells. To evaluate the biologicalactivity of CCL-21 in the vault, T2 hybridoma cells were used thatconstitutively express CCR7. Two different concentrations of CCL-21vault complexes (200 ng and 600 ng), empty vaults (600 ng), andrecombinant CCL-21 (600 ng) were placed in the bottom chamber of a24-well transwell plates and 2×10⁵ T2 cells were loaded in the upperchamber.

In FIG. 2A, the number of cells that migrated to the lower chamberfollowing incubation was determined by flow cytometry and represented asthe % migration. 2.0×10⁵ T2 cells were plated in serum-free medium inthe upper chamber. CCL-21-mCherry-vault complexes (200 ng/ml or 600ng/ml), recombinant CCL-21 (600 ng/ml), control vaults (600 ng/ml) orneutralizing anti-CCL-21 recombinant antibody (5 μg/ml) were added tothe lower chamber of the wells. Following a two hour incubation,migration of T2 cells were analyzed by flow cytometry. CCL-21 vaultcomplexes effectively increased the T2 migration as compared withcontrol, and anti-CCL-21 neutralizing Ab. abrogated the increase inmigration suggesting that CCL-21 vault complexes are biological activeand can mediate the chemotatic migration of T cells. Data in the panelare representative of 2 independent experiments. (Bars; Mean±SEM,*p<0.05 between the CCL-21 vault complexes and control vault oranti-CCL-21 antibody treatment groups.)

More than 7.5% of the T2 cells responded to 200 ng of CCL-21 vaultcomplexes compared with ≦2.5% of the T2 cells incubated with 600 ng ofrecombinant CCL-21. This is a phenomenal response considering that thegiven concentration is of CCL-vault complexes and the actualconcentration of CCL-21 inside of the vaults would be estimated to be≦20 ng. It is possible that the increased bioactivity of CCL-21 vaultcomplexes results from increased stabilization of CCL-21 resulting frompackaging of the protein into the protective environment of the vaultlumen. As the fusion protein non-covalently associates within vaults, itis plausible that vault breathing in solution releases CCL-21 in agradient fashion and the number of cells migrated was higher than therecombinant CCL-21 because a steeper gradient is formed. To demonstratethat the migration of T2 cells was CCL-21 dependent, a neutralizingantibody (against CCL-21) was shown to efficiently block the chemotacticactivity of both recombinant CCL-21 and CCL-21 vault complexes. This ledto the conclusion that CCL-21 vault complexes were functionally activeat inducing the migration of T2 cells in vitro.

These results demonstrate that CCL-21 cytokines retain their biologicalfunction when packaged inside the vault complex.

Example 3 CCL-21 Vault Complexes Enhance DC APC Activity

In order to determine the effect of CCL-21 vault complexes on dendriticcell (DC) antigen presenting cell (APC) activity, the impact ofCCL-21-vault complexes on DC APC activity was studied in vitro. Incomparison to control vaults, CCL-21-vault complexes augmented DCcapacity to process and present ovalbumin and activate CD8 T cells tosecrete IL-2 (FIG. 2B).

FIG. 2B shows CCL-21 vault complexes enhanced DC APC activity, andblocking CCL-21 reversed the increase in APC activity. B3Z cells (1×10⁵cells/200 ul/well) were co-cultured with DC 2.4 (5×10⁴ cells/200ul/well) and ovalbumin (350 ug/ml) in the presence or absence of CCL21vaults (200 ng/ml) and anti-CCL-21 antibody (5 ug/ml) or controlantibody (5 ug/ml goat IgG) for 24 hrs. Control vaults were used atconcentration of 200 ng/ml. T cell activation was analyzed by measuringIL-2 production by ELISA. Data are representative of 2 independentexperiments. (Bars; Mean±SEM, *p<0.05 between the CCL21 vault andcontrol vault or anti-CCL21 antibody treatment groups.) Neutralizationof CCL-21 abrogated the increase in DC APC activity to control levels.

These results demonstrate that CCL-21 vault complexes enhance DC APCactivity in vitro.

Example 4 CCL-21 Vault Complexes Enhance the Recruitment of AntitumorLeukocytic Infiltrates and Reduce 3LL Tumor Burden In Vivo

To determine the anti-tumor activity of CCL-21 vault complexes in vivo,CCL-21 vault complexes were tested for effects on established tumorburden in 3LL tumor-bearing mice.

As shown in FIG. 3, a single intratumoral injection of CCL-21 vaultcomplexes (200 ng) led to significant decrease in tumor burden comparedto empty vaults. C57BL/6 mice (n=5) were injected s.c. with 1.5×10⁵ 3LLtumor cells and tumor growth was monitored daily. After 5 days followingtumor implantation, mice were treated with vaults containingCCL-21-mCherry-mINT (200 ng), vaults alone (200 ng) or normal saline(diluent) via intra tumoral injection and tumor growth was monitored forthe duration of the experiment. Tumor size was measured and tumor volumecalculated as described herein. Bisecting tumor diameters were measuredwith calipers. Intra-tumoral administration of vaults containingCCL-21-mCherry-mINT led to significant reduction in tumor volumecompared with untreated tumor bearing mice (p<0.001).

FIGS. 4A-4B show that the CCL-21-vault complex treatment group (FIG. 4B)had enhanced leukocytic infiltrates compared to control vault (FIG. 4A),and immune staining showed that the infiltrates were mostly CD3expressing T cells (FIG. 4B, bottom right panel). Sections fromparaffin-embedded tissue were stained by hematoxylin-eosin (H&E) andCD3, CD4, CD8 and S-100 by immunohistochemistry using commerciallyavailable antibodies. Each panel of FIGS. 4A and 4B show photographstaken at 400× of representative areas within distinct primary tumors andLymph nodes. Thus, CCL-21 vaults reduced tumor burden and increased theinflux of CD3 expressing T cells in the tumor as compared to controlvaults. (Data; Mean±SEM, *p<0.05 between CCL-21 vaults and controlgroup, n=8 mice/group.)

In additional experiments, the antitumor efficacy of CCL-21-vaultcomplexes was determined in a 7-day established orthotopic 3LL lungcancer model. CCL-21-vault complexes reduced tumor burden by 7-foldcompared to controls. In FIGS. 5A-5C, H&E staining of lung tumorsections from diluent or control vaults showed increased tumor masses ascompared to reduced tumor mass in the CCL-21 vault complex treatmentgroup. FIG. 5A illustrates the effects of treatment of 3LL lung cancercells with diluents only. FIG. 5B shows treatment with control vault,and FIG. 5C shows treatment with CCL-21 vault.

FIG. 6A shows the percentage tumor burden in naïve cells, and 3LL cellstreated with diluents, control vault (CV), or CCL-21 vault complexes ina flow cytometry assay. Tumor burden was calculated on total percentageof GFP and Epcam expressing tumor cells in total lung digest. Naïve lungwas used as control to set up the gate. CCL-21 vault complex treatmentreduced tumor burden. FIG. 6B is a graph illustrating the percentagetumor burden in 3LL cells after treatment with diluents, control vault,or CCL-21 vault complexes.

Example 5 CCL-21-Vault Complexes Induce Tumor Infiltrating T Cell IFNγbut Reduce IL-10 and Augment Systemic T Cell Cytolytic Activity

The effect of CCL-21 vault complexes on inducing tumor infiltrates andIL-10 expression was studied in vivo.

Accompanying the reduced tumor burden, an evaluation of intratumoralleukocytic populations showed enhanced frequency of CD4, CD8, CD3 CXCR3,CD3 CCR7 and DEC205 but reduced levels of MDSC and Tregs (FIG. 7A-7D).In FIG. 7A, CCL-21 vault complexes augmented CD4, CD8, CXCR3⁺CD3⁺T,CCR7′CD3⁺T and DEC205⁺DC infiltrates and reduced MDSC and Tregs comparedwith treatment with diluents or control vault alone. FIGS. 7B and 7Cshow T cell infiltrates of CCL-21 vault complex treatment mice increasedIFNγ and reduced IL-10 expression compared to controls. FIG. 7Dillustrates that CCL-21 vault treatment enhanced the cytolytic activityof purified splenic T cells against parental 3LL tumors in vitro (E:T of20:1 and 40:1).

FIGS. 6A-7D demonstrate that CCL-21 vaults reduced tumor burden andimmune suppressors, increased T and DC infiltrates and induced systemicT cell antitumor activity. Specifically, CCL-21 vaults reduced tumorburden and immune suppressors (MDSC and Tregs) and enhanced intratumoralimmune cell infiltrates in a 3LL orthotropic lung cancer model. Forexperiments shown in FIGS. 6A-7D, 5×10 ³ 3LL-GFP tumor cells wereinjected in the left lung via transthoracic route. One week after tumorinjections, mice were treated with diluent (NS), control vaults (2 ug)or CCL-21 vaults (2 ug) via transthoracic route in the left lung. Day 28post tumor implantation, lung tumors were harvested for the analysis oftumor burden and tumor leukocytic infiltrates. (Data bars, mean±SEM,*p<0.05 between CCL-21 vaults and control vault groups, n=8mice/group.).

These results demonstrate that CCL-21 vault complexes induce tumorinfiltrating T cell IFNγ but reduce IL-10 and augment systemic T cellcytolytic activity.

Example 6 Use of CCL-21 Vault Complexes for Treatment of Cancer inHumans

For treatment of cancer in humans, the pharmaceutical compositions usedin the present invention may be administered in a number of waysdepending upon the invasiveness of treatment and based on whether localor systemic treatment is desired. The preferred initial treatment may beperformed by intra-tumoral injection of the CCL-21 vault complex into atumor of the patient. In some embodiments, intra-tumoral injection of aCCL-21 vault complex is performed on a tumor in the lung of the patientin need of treatment of lung cancer.

In certain embodiments, various dosages of the pharmaceuticalcomposition comprising CCL-21 vault complexes can be administered to thepatient.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

TABLE 1 Sequences Mouse CCL-21 Protein sequence SEQ ID NO: 1MAQMMTLSLLSLVLALCIPWTQGSDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFSPRKHSKPELCANPEEGWVQNLMRRLDQFPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG Human CCL-21 Protein sequence SEQ ID NO: 2MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGP Mouse CCL-21 DNA cloned sequence SEQ ID NO: 3ATGGCTCAGATGATGACTCTGAGCCTCCTTAGCCTGGTCCTGGCTCTCTGCATCCCCTGGACCCAAGGCAGTGATGGAGGGGGTCAGGACTGCTGCCTTAAGTACAGCCAGAAGAAAATTCCCTACAGTATTGTCCGAGGCTATAGGAAGCAAGAACCAAGTTTAGGCTGTCCCATCCCGGCAATCCTGTTCTCACCCCGGAAGCACTCTAAGCCTGAGCTATGTGCAAACCCTGAGGAAGGCTGGGTGCAGAACCTGATGCGCCGCCTGGACCAGCCTCCAGCCCCAGGGAAACAAAGCCCCGGCTGCAGGAAGAACCGGGGAACCTCTAAGTCTGGAAAGAAAGGAAAGGGCTCCAAGGGCTGCAAGAGAACTGAACAGACACAGCCCTCAAGAGGAMouse CCL-21 DNA full sequence SEQ ID NO: 4ATGGCTCAGATGATGACTCTGAGCCTCCTTAGCCTGGTCCTGGCTCTCTGCATCCCCTGGACCCAAGGCAGTGATGGAGGGGGTCAGGACTGCTGCCTTAAGTACAGCCAGAAGAAAATTCCCTACAGTATTGTCCGAGGCTATAGGAAGCAAGAACCAAGTTTAGGCTGTCCCATCCCGGCAATCCTGTTCTCACCCCGGAAGCACTCTAAGCCTGAGCTATGTGCAAACCCTGAGGAAGGCTGGGTGCAGAACCTGATGCGCCGCCTGGACCAGCCTCCAGCCCCAGGGAAACAAAGCCCCGGCTGCAGGAAGAACCGGGGAACCTCTAAGTCTGGAAAGAAAGGAAAGGGCTCCAAGGGCTGCAAGAGAACTGAACAGACACAGCCCTCAAGAGGATAGHuman CCL-21 DNA sequence, Genbank #NP_002980 SEQ ID NO: 5ATGGCTCAGTCACTGGCTCTGAGCCTCCTTATCCTGGTTCTGGCCTTTGGCATCCCCAGGACCCAAGGCAGTGATGGAGGGGCTCAGGACTGTTGCCTCAAGTACAGCCAAAGGAAGATTCCCGCCAAGGTTGTCCGCAGCTACCGGAAGCAGGAACCAAGCTTAGGCTGCTCCATCCCAGCTATCCTGTTCTTGCCCCGCAAGCGCTCTCAGGCAGAGCTATGTGCAGACCCAAAGGAGCTCTGGGTGCAGCAGCTGATGCAGCATCTGGACAAGACACCATCCCCACAGAAACCAGCCCAGGGCTGCAGGAAGGACAGGGGGGCCTCCAAGACTGGCAAGAAAGGAAAGGGCTCCAAAGGCTGCAAGAGGACTGAGCGGTCACAGACCCCTAAAGGGCCATAGMouse mINT DNA sequence SEQ ID NO: 6TGC ACA CAA CAC TGG CAG GAT GCT GTG CCT TGG ACA GAA CTC CTC AGT CTA CAG ACA GAG GATGGC TTC TGG AAA CTT ACA CCA GAA CTG GGA CTT ATA TTA AAT CTT AAT ACA AAT GGT TTG CACAGC TTT CTT AAA CAA AAA GGC ATT CAA TCT CTA GGT GTA AAA GGA AGA GAA TGT CTC CTG GACCTA ATT GCC ACA ATG CTG GTA CTA CAG TTT ATT CGC ACC AGG TTG GAA AAA GAG GGA ATA GTGTTC AAA TCA CTG ATG AAA ATG GAT GAC CCT TCT ATT TCC AGG AAT ATT CCC TGG GCT TTT GAGGCA ATA AAG CAA GCA AGT GAA TGG GTA AGA AGA ACT GAA GGA CAG TAC CCA TCT ATC TGC CCACGG CTT GAA CTG GGG AAC GAC TGG GAC TCT GCC ACC AAG CAG TTG CTG GGA CTC CAG CCC ATAAGC ACT GTG TCC CCT CTT CAT AGA GTC CTC CAT TAC AGT CAA GGC TAAHuman mINT DNA sequence SEQ ID NO: 7TGC ACA CAA CAC TGG CAG GAT GCT GTG CCT TGG ACA GAA CTC CTC AGT CTA CAG ACA GAG GATGGC TTC TGG AAA CTT ACA CCA GAA CTG GGA CTT ATA TTA AAT CTT AAT ACA AAT GGT TTG CACAGC TTT CTT AAA CAA AAA GGC ATT CAA TCT CTA GGT GTA AAA GGA AGA GAA TGT CTC CTG GACCTA ATT GCC ACA ATG CTG GTA CTA CAG TTT ATT CGC ACC AGG TTG GAA AAA GAG GGA ATA GTGTTC AAA TCA CTG ATG AAA ATG GAT GAC CCT TCT ATT TCC AGG AAT ATT CCC TGG GCT TTT GAGGCA ATA AAG CAA GCA AGT GAA TGG GTA AGA AGA ACT GAA GGA CAG TAC CCA TCT ATC TGC CCACGG CTT GAA CTG GGG AAC GAC TGG GAC TCT GCC ACC AAG CAG TTG CTG GGA CTC CAG CCC ATAAGC ACT GTG TCC CCT CTT CAT AGA GTC CTC CAT TAC AGT CAA GGC TAAHuman mINT protein sequence (residues 1563-1724 of the human VPARP proteinsequence) SEQ ID NO: 8ctqhwqdavpwtells1qtedgfwkltpelglilnlntnglhsflkqkgicislgvkgreclldliatmlvlqfirtrlekegivfkslmkmddpsisrnipwafeaikqasewvrrtegqypsicprlelgndwdsatkqllglqpistvsplhrylhysqgMouse mINT protein sequence SEQ ID NO: 9CTQHWQDAVPWTELLSLQTEDGFWKLTPELGLILNLNTNGLHSFLKQKGIQSLGVKGRECLLDLIATMLVLQFIRTRLEKEGIVFKSLMKMDDPSISRNIPWAFEAIKQASEWVRRTEGQYPSICPRLELGNDWDSATKQLLGLQPISTVSPLHRVLHYSQGMouse CCL-21-mINT fusion DNA sequence SEQ ID NO: 10ATGGCTCAGATGATGACTCTGAGCCTCCTTAGCCTGGTCCTGGCTCTCTGCATCCCCTGGACCCAAGGCAGTGATGGAGGGGGTCAGGACTGCTGCCTTAAGTACAGCCAGAAGAAAATTCCCTACAGTATTGTCCGAGGCTATAGGAAGCAAGAACCAAGTTTAGGCTGTCCCATCCCGGCAATCCTGTTCTCACCCCGGAAGCACTCTAAGCCTGAGCTATGTGCAAACCCTGAGGAAGGCTGGGTGCAGAACCTGATGCGCCGCCTGGACCAGCCTCCAGCCCCAGGGAAACAAAGCCCCGGCTGCAGGAAGAACCGGGGAACCTCTAAGTCTGGAAAGAAAGGAAAGGGCTCCAAGGGCTGCAAGAGAACTGAACAGACACAGCCCTCAAGAGGA TGC ACA CAA CAC TGG CAG GAT GCTGTG CCT TGG ACA GAA CTC CTC AGT CTA CAG ACA GAG GAT GGC TTC TGG AAA CTT ACA CCA GAACTG GGA CTT ATA TTA AAT CTT AAT ACA AAT GGT TTG CAC AGC TTT CTT AAA CAA AAA GGC ATTCAA TCT CTA GGT GTA AAA GGA AGA GAA TGT CTC CTG GAC CTA ATT GCC ACA ATG CTG GTA CTACAG TTT ATT CGC ACC AGG TTG GAA AAA GAG GGA ATA GTG TTC AAA TCA CTG ATG AAA ATG GATGAC CCT TCT ATT TCC AGG AAT ATT CCC TGG GCT TTT GAG GCA ATA AAG CAA GCA AGT GAA TGGGTA AGA AGA ACT GAA GGA CAG TAC CCA TCT ATC TGC CCA CGG CTT GAA CTG GGG AAC GAC TGGGAC TCT GCC ACC AAG CAG TTG CTG GGA CTC CAG CCC ATA AGC ACT GTG TCC CCT CTT CAT AGAGTC CTC CAT TAC AGT CAA GGC TAA Human CCL-21-mINT fusion DNA sequenceSEQ ID NO: 11ATGGCTCAGTCACTGGCTCTGAGCCTCCTTATCCTGGTTCTGGCCTTTGGCATCCCCAGGACCCAAGGCAGTGATGGAGGGGCTCAGGACTGTTGCCTCAAGTACAGCCAAAGGAAGATTCCCGCCAAGGTTGTCCGCAGCTACCGGAAGCAGGAACCAAGCTTAGGCTGCTCCATCCCAGCTATCCTGTTCTTGCCCCGCAAGCGCTCTCAGGCAGAGCTATGTGCAGACCCAAAGGAGCTCTGGGTGCAGCAGCTGATGCAGCATCTGGACAAGACACCATCCCCACAGAAACCAGCCCAGGGCTGCAGGAAGGACAGGGGGGCCTCCAAGACTGGCAAGAAAGGAAAGGGCTCCAAAGGCTGCAAGAGGACTGAGCGGTCACAGACCCCTAAAGGGCCAGCTAGCTGC ACA CAA CAC TGG CAGGAT GCT GTG CCT TGG ACA GAA CTC CTC AGT CTA CAG ACA GAG GAT GGC TTC TGG AAA CTT ACACCA GAA CTG GGA CTT ATA TTA AAT CTT AAT ACA AAT GGT TTG CAC AGC TTT CTT AAA CAA AAAGGC ATT CAA TCT CTA GGT GTA AAA GGA AGA GAA TGT CTC CTG GAC CTA ATT GCC ACA ATG CTGGTA CTA CAG TTT ATT CGC ACC AGG TTG GAA AAA GAG GGA ATA GTG TTC AAA TCA CTG ATG AAAATG GAT GAC CCT TCT ATT TCC AGG AAT ATT CCC TGG GCT TTT GAG GCA ATA AAG CAA GCA AGTGAA TGG GTA AGA AGA ACT GAA GGA CAG TAC CCA TCT ATC TGC CCA CGG CTT GAA CTG GGG AACGAC TGG GAC TCT GCC ACC AAG CAG TTG CTG GGA CTC CAG CCC ATA AGC ACT GTG TCC CCT CTTCAT AGA GTC CTC CAT TAC AGT CAA GGC TAAMouse CCL-21-INT fusion Protein Sequence SEQ ID NO: 12MAQMMTLSLLSLVLALCIPWTQGSDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFSPRKHSKPELCANPEEGWVQNLMRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRGCTQHWQDAVPWTELLSLQTEDGFWKLTPELGLILNLNTNGLHSFLKQKGIQSLGVKGRECLLDLIATMLVLQFIRTRLEKEGIVFKSLMKMDDPSISRNIPWAFEAIKQASEWVRRTEGQYPSICPRLELGNDWDSATKQLLGLQPISTVSPLHRVLHYSQGHuman CCL-21-INT fusion Protein Sequence SEQ ID NO: 13MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGPASCTQHWQDAVPWTELLSLQTEDGFWKLTPELGLILNLNTNGLHSFLKQKGIQSLGVKGRECLLDLIATMLVLQFIRTRLEKEGIVFKSLMKMDDPSISRNIPWAFEAIKQASEWVRRTEGQYPSICPRLELGNDWDSATKQLLGLQPISTVSPLHRVLHYSQGHuman VPARP protein sequence Genbank #AAD47250 SEQ ID NO: 14Met Val Met Gly Ile Phe Ala Asn Cys Ile Phe Cys Leu Lys Val LysTyr Leu Pro Gln Gln Gln Lys Lys Lys Leu Gln Thr Asp Ile Lys GluAsn Gly Gly Lys Phe Ser Phe Ser Leu Asn Pro Gln Cys Thr His IleIle Leu Asp Asn Ala Asp Val Leu Ser Gln Tyr Gln Leu Asn Ser IleGln Lys Asn His Val His Ile Ala Asn Pro Asp Phe Ile Trp Lys SerIle Arg Glu Lys Arg Leu Leu Asp Val Lys Asn Tyr Asp Pro Tyr LysPro Leu Asp Ile Thr Pro Pro Pro Asp Gln Lys Ala Ser Ser Ser GluVal Lys Thr Glu Gly Leu Cys Pro Asp Ser Ala Thr Glu Glu Glu AspThr Val Glu Leu Thr Glu Phe Gly Met Gln Asn Val Glu Ile Pro HisLeu Pro Gln Asp Phe Glu Val Ala Lys Tyr Asn Thr Leu Glu Lys ValGly Met Glu Gly Gly Gln Glu Ala Val Val Val Glu Leu Gln Cys SerArg Asp Ser Arg Asp Cys Pro Phe Leu Ile Ser Ser His Phe Leu LeuAsp Asp Gly Met Glu Thr Arg Arg Gln Phe Ala Ile Lys Lys Thr SerGlu Asp Ala Ser Glu Tyr Phe Glu Asn Tyr Ile Glu Glu Leu Lys LysGln Gly Phe Leu Leu Arg Glu His Phe Thr Pro Glu Ala Thr Gln LeuAla Ser Glu Gln Leu Gln Ala Leu Leu Leu Glu Glu Val Met Asn SerSer Thr Leu Ser Gln Glu Val Ser Asp Leu Val Glu Met Ile Trp AlaGlu Ala Leu Gly His Leu Glu His Met Leu Leu Lys Pro Val Asn ArgIle Ser Leu Asn Asp Val Ser Lys Ala Glu Gly Ile Leu Leu Leu ValLys Ala Ala Leu Lys Asn Gly Glu Thr Ala Glu Gln Leu Gln Lys MetMet Thr Glu Phe Tyr Arg Leu Ile Pro His Lys Gly Thr Met Pro LysGlu Val Asn Leu Gly Leu Leu Ala Lys Lys Ala Asp Leu Cys Gln LeuIle Arg Asp Met Val Asn Val Cys Glu Thr Asn Leu Ser Lys Pro AsnPro Pro Ser Leu Ala Lys Tyr Arg Ala Leu Arg Cys Lys Ile Glu HisVal Glu Gln Asn Thr Glu Glu Phe Leu Arg Val Arg Lys Glu Val LeuGln Asn His His Ser Lys Ser Pro Val Asp Val Leu Gln Ile Phe ArgVal Gly Arg Val Asn Glu Thr Thr Glu Phe Leu Ser Lys Leu Gly AsnVal Arg Pro Leu Leu His Gly Ser Pro Val Gln Asn Ile Val Gly IleLeu Cys Arg Gly Leu Leu Leu Pro Lys Val Val Glu Asp Arg Gly ValGln Arg Thr Asp Val Gly Asn Leu Gly Ser Gly Ile Tyr Phe Ser AspSer Leu Ser Thr Ser Ile Lys Tyr Ser His Pro Gly Glu Thr Asp GlyThr Arg Leu Leu Leu Ile Cys Asp Val Ala Leu Gly Lys Cys Met AspLeu His Glu Lys Asp Phe Pro Leu Thr Glu Ala Pro Pro Gly Tyr AspSer Val His Gly Val Ser Gln Thr Ala Ser Val Thr Thr Asp Phe GluAsp Asp Glu Phe Val Val Tyr Lys Thr Asn Gln Val Lys Met Lys TyrIle Ile Lys Phe Ser Met Pro Gly Asp Gln Ile Lys Asp Phe His ProSer Asp His Thr Glu Leu Glu Glu Tyr Arg Pro Glu Phe Ser Asn PheSer Lys Val Glu Asp Tyr Gln Leu Pro Asp Ala Lys Thr Ser Ser SerThr Lys Ala Gly Leu Gln Asp Ala Ser Gly Asn Leu Val Pro Leu GluAsp Val His Ile Lys Gly Arg Ile Ile Asp Thr Val Ala Gln Val IleVal Phe Gln Thr Tyr Thr Asn Lys Ser His Val Pro Ile Glu Ala LysTyr Ile Phe Pro Leu Asp Asp Lys Ala Ala Val Cys Gly Phe Glu AlaPhe Ile Asn Gly Lys His Ile Val Gly Glu Ile Lys Glu Lys Glu GluAla Gln Gln Glu Tyr Leu Glu Ala Val Thr Gln Gly His Gly Ala TyrLeu Met Ser Gln Asp Ala Pro Asp Val Phe Thr Val Ser Val Gly AsnLeu Pro Pro Lys Ala Lys Val Leu Ile Lys Ile Thr Tyr Ile Thr GluLeu Ser Ile Leu Gly Thr Val Gly Val Phe Phe Met Pro Ala Thr ValAla Pro Trp Gln Gln Asp Lys Ala Leu Asn Glu Asn Leu Gln Asp ThrVal Glu Lys Ile Cys Ile Lys Glu Ile Gly Thr Lys Gln Ser Phe SerLeu Thr Met Ser Ile Glu Met Pro Tyr Val Ile Glu Phe Ile Phe SerAsp Thr His Glu Leu Lys Gln Lys Arg Thr Asp Cys Lys Ala Val IleSer Thr Met Glu Gly Ser Ser Leu Asp Ser Ser Gly Phe Ser Leu HisIle Gly Leu Ser Ala Ala Tyr Leu Pro Arg Met Trp Val Glu Lys HisPro Glu Lys Glu Ser Glu Ala Cys Met Leu Val Phe Gln Pro Asp LeuAsp Val Asp Leu Pro Asp Leu Ala Ser Glu Ser Glu Val Ile Ile CysLeu Asp Cys Ser Ser Ser Met Glu Gly Val Thr Phe Leu Gln Ala LysGln Ile Thr Leu His Ala Leu Ser Leu Val Gly Glu Lys Gln Lys ValAsn Ile Ile Gln Phe Gly Thr Gly Tyr Lys Glu Leu Phe Ser Tyr ProLys His Ile Thr Ser Asn Thr Thr Ala Ala Glu Phe Ile Met Ser AlaThr Pro Thr Met Gly Asn Thr Asp Phe Trp Lys Thr Leu Arg Tyr LeuSer Leu Leu Tyr Pro Ala Arg Gly Ser Arg Asn Ile Leu Leu Val SerAsp Gly His Leu Gln Asp Glu Ser Leu Thr Leu Gln Leu Val Lys ArgSer Arg Pro His Thr Arg Leu Phe Ala Cys Gly Ile Gly Ser Thr AlaAsn Arg His Val Leu Arg Ile Leu Ser Gln Cys Gly Ala Gly ValPhe Glu Tyr Phe Asn Ala Lys Ser Lys His Ser Trp Arg Lys GlnIle Glu Asp Gln Met Thr Arg Leu Cys Ser Pro Ser Cys His SerVal Ser Val Lys Trp Gln Gln Leu Asn Pro Asp Ala Pro Glu AlaLeu Gln Ala Pro Ala Gln Val Pro Ser Leu Phe Arg Asn Asp ArgLeu Leu Val Tyr Gly Phe Ile Pro His Cys Thr Gln Ala Thr LeuCys Ala Leu Ile Gln Glu Lys Glu Phe Cys Thr Met Val Ser ThrThr Glu Leu Gln Lys Thr Thr Gly Thr Met Ile His Lys Leu AlaAla Arg Ala Leu Ile Arg Asp Tyr Glu Asp Gly Ile Leu His GluAsn Glu Thr Ser His Glu Met Lys Lys Gln Thr Leu Lys Ser LeuIle Ile Lys Leu Ser Lys Glu Asn Ser Leu Ile Thr Gln Phe ThrSer Phe Val Ala Val Glu Lys Arg Asp Glu Asn Glu Ser Pro PhePro Asp Ile Pro Lys Val Ser Glu Leu Ile Ala Lys Glu Asp ValAsp Phe Leu Pro Tyr Met Ser Trp Gln Gly Glu Pro Gln Glu AlaVal Arg Asn Gln Ser Leu Leu Ala Ser Ser Glu Trp Pro Glu LeuArg Leu Ser Lys Arg Lys His Arg Lys Ile Pro Phe Ser Lys ArgLys Met Glu Leu Ser Gln Pro Glu Val Ser Glu Asp Phe Glu GluAsp Gly Leu Gly Val Leu Pro Ala Phe Thr Ser Asn Leu Glu ArgGly Gly Val Glu Lys Leu Leu Asp Leu Ser Trp Thr Glu Ser CysLys Pro Thr Ala Thr Glu Pro Leu Phe Lys Lys Val Ser Pro TrpGlu Thr Ser Thr Ser Ser Phe Phe Pro Ile Leu Ala Pro Ala ValGly Ser Tyr Leu Thr Pro Thr Thr Arg Ala His Ser Pro Ala SerLeu Ser Phe Ala Ser Tyr Arg Gln Val Ala Ser Phe Gly Ser AlaAla Pro Pro Arg Gln Phe Asp Ala Ser Gln Phe Ser Gln Gly ProVal Pro Gly Thr Cys Ala Asp Trp Ile Pro Gln Ser Ala Ser CysPro Thr Gly Pro Pro Gln Asn Pro Pro Ser Ala Pro Tyr Cys GlyIle Val Phe Ser Gly Ser Ser Leu Ser Ser Ala Gln Ser Ala ProLeu Gln His Pro Gly Gly Phe Thr Thr Arg Pro Ser Ala Gly ThrPhe Pro Glu Leu Asp Ser Pro Gln Leu His Phe Ser Leu Pro ThrAsp Pro Asp Pro Ile Arg Gly Phe Gly Ser Tyr His Pro Ser AlaTyr Ser Pro Phe His Phe Gln Pro Ser Ala Ala Ser Leu Thr AlaAsn Leu Arg Leu Pro Met Ala Ser Ala Leu Pro Glu Ala Leu CysSer Gln Ser Arg Thr Thr Pro Val Asp Leu Cys Leu Leu Glu GluSer Val Gly Ser Leu Glu Gly Ser Arg Cys Pro Val Phe Ala PheGln Ser Ser Asp Thr Glu Ser Asp Glu Leu Ser Glu Val Leu GlnAsp Ser Cys Phe Leu Gln Ile Lys Cys Asp Thr Lys Asp Asp SerIle Pro Cys Phe Leu Glu Leu Lys Glu Glu Asp Glu Ile Val CysThr Gln His Trp Gln Asp Ala Val Pro Trp Thr Glu Leu Leu SerLeu Gln Thr Glu Asp Gly Phe Trp Lys Leu Thr Pro Glu Leu GlyLeu Ile Leu Asn Leu Asn Thr Asn Gly Leu His Ser Phe Leu LysGln Lys Gly Ile Gln Ser Leu Gly Val Lys Gly Arg Glu Cys LeuLeu Asp Leu Ile Ala Thr Met Leu Val Leu Gln Phe Ile Arg ThrArg Leu Glu Lys Glu Gly Ile Val Phe Lys Ser Leu Met Lys MetAsp Asp Pro Ser Ile Ser Arg Asn Ile Pro Trp Ala Phe Glu AlaIle Lys Gln Ala Ser Glu Trp Val Arg Arg Thr Glu Gly Gln TyrPro Ser Ile Cys Pro Arg Leu Glu Leu Gly Asn Asp Trp Asp SerAla Thr Lys Gln Leu Leu Gly Leu Gln Pro Ile Ser Thr Val SerPro Leu His Arg Val Leu His Tyr Ser Gln GlyHuman VPARP cDNA, Genbank #AF158255 SEQ ID NO: 15atggtgatgg gaatctttgc aaattgtatc ttctgtttga aagtgaagta cttacctcagcagcagaaga aaaagctaca aactgacatt aaggaaaatg gcggaaagtt ttccttttcgttaaatcctc agtgcacaca tataatctta gataatgctg atgttctgag tcagtaccaactgaattcta tccaaaagaa ccacgttcat attgcaaacc cagattttat atggaaatctatcagagaaa agagactctt ggatgtaaag aattatgatc cttataagcc cctggacatcacaccacctc ctgatcagaa ggcgagcagt tctgaagtga aaacagaagg tctatgcccggacagtgcca cagaggagga agacactgtg gaactcactg agtttggtat gcagaatgttgaaattcctc atcttcctca agattttgaa gttgcaaaat ataacacctt ggagaaagtgggaatggagg gaggccagga agctgtggtg gtggagcttc agtgttcgcg ggactccagggactgtcctt tcctgatatc ctcacacttc ctcctggatg atggcatgga gactagaagacagtttgcta taaagaaaac ctctgaagat gcaagtgaat actttgaaaa ttacattgaagaactgaaga aacaaggatt tctactaaga gaacatttca cacctgaagc aacccaattagcatctgaac aattgcaagc attgcttttg gaggaagtca tgaattcaag cactctgagccaagaggtga gcgatttagt agagatgatt tgggcagagg ccctgggcca cctggaacacatgcttctca agccagtgaa caggattagc ctcaacgatg tgagcaaggc agaggggattctccttctag taaaggcagc actgaaaaat ggagaaacag cagagcaatt gcaaaagatgatgacagagt tttacagact gatacctcac aaaggcacaa tgcccaaaga agtgaacctgggactattgg ctaagaaagc agacctctgc cagctaataa gagacatggt taatgtctgtgaaactaatt tgtccaaacc caacccacca tccctggcca aataccgagc tttgaggtgcaaaattgagc atgttgaaca gaatactgaa gaatttctca gggttagaaa agaggttttgcagaatcatc acagtaagag cccagtggat gtcttgcaga tatttagagt tggcagagtgaatgaaacca cagagttttt gagcaaactt ggtaatgtga ggcccttgtt gcatggttctcctgtacaaa acatcgtggg aatcttgtgt cgagggttgc ttttacccaa agtagtggaagatcgtggtg tgcaaagaac agacgtcgga aaccttggaa gtgggattta tttcagtgattcgctcagta caagtatcaa gtactcacac ccgggagaga cagatggcac cagactcctgctcatttgtg acgtagccct cggaaagtgt atggacttac atgagaagga ctttcccttaactgaagcac caccaggcta cgacagtgtg catggagttt cacaaacagc ctctgtcaccacagactttg aggatgatga atttgttgtc tataaaacca atcaggttaa aatgaaatatattattaaat tttccatgcc tggagatcag ataaaggact ttcatcctag tgatcatactgaattagagg aatacagacc tgagttttca aatttttcaa aggttgaaga ttaccagttaccagatgcca aaacttccag cagcaccaag gccggcctcc aggatgcctc tgggaacttggttcctctgg aggatgtcca catcaaaggg agaatcatag acactgtagc ccaggtcattgtttttcaga catacacaaa taaaagtcac gtgcccattg aggcaaaata tatctttcctttggatgaca aggccgctgt gtgtggcttc gaagccttca tcaatgggaa gcacatagttggagagatta aagagaagga agaagcccag caagagtacc tagaagccgt gacccagggccatggcgctt acctgatgag tcaggatgct ccggacgttt ttactgtaag tgttggaaacttacccccta aggctaaggt tcttataaaa attacctaca tcacagaact cagcatcctgggcactgttg gtgtcttttt catgcccgcc accgtagcac cctggcaaca ggacaaggctttgaatgaaa accttcagga tacagtagag aagatttgta taaaagaaat aggaacaaagcaaagcttct ctttgactat gtctattgag atgccgtatg tgattgaatt cattttcagtgatacacatg aactgaaaca aaagcgcaca gactgcaaag ctgtcattag caccatggaaggcagctcct tagacagcag tggattttct ctccacatcg gtttgtctgc tgcctatctcccaagaatgt gggttgaaaa acatccagaa aaagaaagcg aggcttgcat gcttgtctttcaacccgatc tcgatgtcga cctccctgac ctagccagtg agagcgaagt gattatttgtcttgactgct ccagttccat ggagggtgtg acattcttgc aagccaagca aatcaccttgcatgcgctgt ccttggtggg tgagaagcag aaagtaaata ttatccagtt cggcacaggttacaaggagc tattttcgta tcctaagcat atcacaagca ataccacggc agcagagttcatcatgtctg ccacacctac catggggaac acagacttct ggaaaacact ccgatatcttagcttattgt accctgctcg agggtcacgg aacatcctcc tggtgtctga tgggcacctccaggatgaga gcctgacatt acagctcgtg aagaggagcc gcccgcacac caggttattcgcctgcggta tcggttctac agcaaatcgt cacgtcttaa ggattttgtc ccagtgtggtgccggagtat ttgaatattt taatgcaaaa tccaagcata gttggagaaa acagatagaagaccaaatga ccaggctatg ttctccgagt tgccactctg tctccgtcaa atggcagcaactcaatccag atgcgcccga ggccctgcag gccccagccc aggtgccatc cttgtttcgcaatgatcgac tccttgtcta tggattcatt cctcactgca cacaagcaac tctgtgtgcactaattcaag agaaagaatt ttgtacaatg gtgtcgacta ctgagcttca gaagacaactggaactatga tccacaagct ggcagcccga gctctaatca gagattatga agatggcattcttcacgaaa atgaaaccag tcatgagatg aaaaaacaaa ccttgaaatc tctgattattaaactcagta aagaaaactc tctcataaca caatttacaa gctttgtggc agttgagaaaagggatgaga atgagtcgcc ttttcctgat attccaaaag tttctgaact tattgccaaagaagatgtag acttcctgcc ctacatgagc tggcaggggg agccccaaga agccgtcaggaaccagtctc ttttagcatc ctctgagtgg ccagaattac gtttatccaa acgaaaacataggaaaattc cattttccaa aagaaaaatg gaattatctc agccagaagt ttctgaagattttgaagagg atggcttagg tgtactacca gctttcacat caaatttgga acgtggaggtgtggaaaagc tattggattt aagttggaca gagtcatgta aaccaacagc aactgaaccactatttaaga aagtcagtcc atgggaaaca tctacttcta gcttttttcc tattttggctccggccgttg gttcctatct taccccgact acccgcgctc acagtcctgc ttccttgtcttttgcctcat atcgtcaggt agctagtttc ggttcagctg ctcctcccag acagtttgatgcatctcaat tcagccaagg ccctgtgcct ggcacttgtg ctgactggat cccacagtcggcgtcttgtc ccacaggacc tccccagaac ccaccttctg caccctattg tggcattgttttttcaggga gctcattaag ctctgcacag tctgctccac tgcaacatcc tggaggctttactaccaggc cttctgctgg caccttccct gagctggatt ctccccagct tcatttctctcttcctacag accctgatcc catcagaggt tttgggtctt atcatccctc tgcttactctccttttcatt ttcaaccttc cgcagcctct ttgactgcca accttaggct gccaatggcctctgctttac ctgaggctct ttgcagtcag tcccggacta ccccagtaga tctctgtcttctagaagaat cagtaggcag tctcgaagga agtcgatgtc ctgtctttgc ttttcaaagttctgacacag aaagtgatga gctatcagaa gtacttcaag acagctgctt tttacaaataaagtgtgata caaaagatga cagtatcccg tgctttctgg aattaaaaga agaggatgaaatagtgtgca cacaacactg gcaggatgct gtgccttgga cagaactcct cagtctacagacagaggatg gcttctggaa acttacacca gaactgggac ttatattaaa tcttaatacaaatggtttgc acagctttct taaacaaaaa ggcattcaat ctctaggtgt aaaaggaagagaatgtctcc tggacctaat tgccacaatg ctggtactac agtttattcg caccaggttggaaaaagagg gaatagtgtt caaatcactg atgaaaatgg atgacccttc tatttccaggaatattccct gggcttttga ggcaataaag caagcaagtg aatgggtaag aagaactgaaggacagtacc catctatctg cccacggctt gaactgggga acgactggga ctctgccaccaagcagttgc tgggactcca gcccataagc actgtgtccc ctcttcatag agtcctccattacagtcaag gctaa Human MVP, Genbank #CAA56256 SEQ ID NO: 16Met Ala Thr Glu Glu Phe Ile Ile Arg Ile Pro Pro Tyr His Tyr IleHis Val Leu Asp Gln Asn Ser Asn Val Ser Arg Val Glu Val Gly ProLys Thr Tyr Ile Arg Gln Asp Asn Glu Arg Val Leu Phe Ala Pro MetArg Met Val Thr Val Pro Pro Arg His Tyr Cys Thr Val Ala Asn ProVal Ser Arg Asp Ala Gln Gly Leu Val Leu Phe Asp Val Thr Gly GlnVal Arg Leu Arg His Ala Asp Leu Glu Ile Arg Leu Ala Gln Asp ProPhe Pro Leu Tyr Pro Gly Glu Val Leu Glu Lys Asp Ile Thr Pro LeuGln Val Val Leu Pro Asn Thr Ala Leu His Leu Lys Ala Leu Leu AspPhe Glu Asp Lys Asp Gly Asp Lys Val Val Ala Gly Asp Glu Trp LeuPhe Glu Gly Pro Gly Thr Tyr Ile Pro Arg Lys Glu Val Glu Val ValGlu Ile Ile Gln Ala Thr Ile Ile Arg Gln Asn Gln Ala Leu Arg LeuArg Ala Arg Lys Glu Cys Trp Asp Arg Asp Gly Lys Glu Arg Val ThrGly Glu Glu Trp Leu Val Thr Thr Val Gly Ala Tyr Leu Pro Ala ValPhe Glu Glu Val Leu Asp Leu Val Asp Ala Val Ile Leu Thr Glu LysThr Ala Leu His Leu Arg Ala Arg Arg Asn Phe Arg Asp Phe Arg GlyVal Ser Arg Arg Thr Gly Glu Glu Trp Leu Val Thr Val Gln Asp ThrGlu Ala His Val Pro Asp Val His Glu Glu Val Leu Gly Val Val ProIle Thr Thr Leu Gly Pro His Asn Tyr Cys Val Ile Leu Asp Pro ValGly Pro Asp Gly Lys Asn Gln Leu Gly Gln Lys Arg Val Val Lys GlyGlu Lys Ser Phe Phe Leu Gln Pro Gly Glu Gln Leu Glu Gln Gly IleGln Asp Val Tyr Val Leu Ser Glu Gln Gln Gly Leu Leu Leu Arg AlaLeu Gln Pro Leu Glu Glu Gly Glu Asp Glu Glu Lys Val Ser His GlnAla Gly Asp His Trp Leu Ile Arg Gly Pro Leu Glu Tyr Val Pro SerAla Lys Val Glu Val Val Glu Glu Arg Gln Ala Ile Pro Leu Asp GluAsn Glu Gly Ile Tyr Val Gln Asp Val Lys Thr Gly Lys Val Arg AlaVal Ile Gly Ser Thr Tyr Met Leu Thr Gln Asp Glu Val Leu Trp GluLys Glu Leu Pro Pro Gly Val Glu Glu Leu Leu Asn Lys Gly Gln AspPro Leu Ala Asp Arg Gly Glu Lys Asp Thr Ala Lys Ser Leu Gln ProLeu Ala Pro Arg Asn Lys Thr Arg Val Val Ser Tyr Arg Val Pro HisAsn Ala Ala Val Gln Val Tyr Asp Tyr Arg Glu Lys Arg Ala Arg ValVal Phe Gly Pro Glu Leu Val Ser Leu Gly Pro Glu Glu Gln Phe ThrVal Leu Ser Leu Ser Ala Gly Arg Pro Lys Arg Pro His Ala Arg ArgAla Leu Cys Leu Leu Leu Gly Pro Asp Phe Phe Thr Asp Val Ile ThrIle Glu Thr Ala Asp His Ala Arg Leu Gln Leu Gln Leu Ala Tyr AsnTrp His Phe Glu Val Asn Asp Arg Lys Asp Pro Gln Glu Thr Ala LysLeu Phe Ser Val Pro Asp Phe Val Gly Asp Ala Cys Lys Ala Ile AlaSer Arg Val Arg Gly Ala Val Ala Ser Val Thr Phe Asp Asp Phe HisLys Asn Ser Ala Arg Ile Ile Arg Thr Ala Val Phe Gly Phe Glu ThrSer Glu Ala Lys Gly Pro Asp Gly Met Ala Leu Pro Arg Pro Arg AspGln Ala Val Phe Pro Gln Asn Gly Leu Val Val Ser Ser Val Asp ValGln Ser Val Glu Pro Val Asp Gln Arg Thr Arg Asp Ala Leu Gln ArgSer Val Gln Leu Ala Ile Glu Ile Thr Thr Asn Ser Gln Glu Ala AlaAla Lys His Glu Ala Gln Arg Leu Glu Gln Glu Ala Arg Gly Arg LeuGlu Arg Gln Lys Ile Leu Asp Gln Ser Glu Ala Glu Lys Ala Arg LysGlu Leu Leu glu Leu Glu Ala Leu Ser Met Ala Val Glu Ser Thr GlyThr Ala Lys Ala Glu Ala Glu Ser Arg Ala Glu Ala Ala Arg Ile GluGly Glu Gly Ser Val Leu Gln Ala Lys Leu Lys Ala Gln Ala Leu AlaIle Glu Thr Glu Ala Glu Leu Gln Arg Val Gln Lys Val Arg Glu LeuGlu Leu Val Tyr Ala Arg Ala Gln Leu Glu Leu Glu Val Ser Lys AlaGln Gln Leu Ala Glu Val Glu Val Lys Lys Phe Lys Gln Met Thr GluAla Ile Gly Pro Ser Thr Ile Arg Asp Leu Ala Val Ala Gly Pro GluMet Gln Val Lys Leu Leu Gln Ser Leu Gly Leu Lys Ser Thr Leu IleThr Asp Gly Ser Thr Pro Ile Asn Leu Phe Asn Thr Ala Phe Gly LeuLeu Gly Met Gly Pro Glu Gly Gln Pro Leu Gly Arg Arg Val Ala SerGly Pro Ser Pro Gly Glu Gly Ile Ser Pro Gln Ser Ala Gln Ala ProGln Ala Pro Gly Asp Asn His Val Val Pro Val Leu ArgHuman MVP cDNA, Genbank #X79882 SEQ ID NO: 17atggcaactg aagagttcat catccgcatc cccccatacc actatatcca tgtgctggaccagaacagca acgtgtcccg tgtggaggtc gggccaaaga cctacatccg gcaggacaatgagagggtac tgtttgcccc catgcgcatg gtgaccgtcc ccccacgtca ctactgcacagtggccaacc ctgtgtctcg ggatgcccag ggcttggtgc tgtttgatgt cacagggcaagttcggcttc gccacgctga cctcgagatc cggctggccc aggacccctt ccccctgtacccaggggagg tgctggaaaa ggacatcaca cccctgcagg tggttctgcc caacactgccctccatctaa aggcgctgct tgattttgag gataaagatg gagacaaggt ggtggcaggagatgagtggc ttttcgaggg acctggcacg tacatccccc ggaaggaagt ggaggtcgtggagatcattc aggccaccat catcaggcag aaccaggcgc tgcggctcag ggcccgcaaggagtgctggg accgggacgg caaggagagg gtgacagggg aagaatggct ggtcaccacagtaggggcgt acctcccagc ggtgtttgag gaggttctgg atttggtgga cgccgtcatccttacggaaa agacagccct gcacctccgg gctcggcgga acttccggga cttcaggggagtgtcccgcc gcactgggga ggagtggctg gtaacagtgc aggacacaga ggcccacgtgccagatgtcc acgaggaggt gctgggggtt gtgcccatca ccaccctggg cccccacaactactgcgtga ttctcgaccc tgtcggaccg gatggcaaga atcagctggg gcagaagcgcgtggtcaagg gagagaagtc ttttttcctc cagccaggag agcagctgga acaaggcatccaggatgtgt atgtgctgtc ggagcagcag gggctgctgc tgagggccct gcagcccctggaggaggggg aggatgagga gaaggtctca caccaggctg gggaccactg gctcatccgcggacccctgg agtatgtgcc atctgccaaa gtggaggtgg tggaggagcg ccaggccatccctctagacg agaacgaggg catctatgtg caggatgtca agaccggaaa ggtgcgcgctgtgattggaa gcacctacat gctgacccag gacgaagtcc tgtgggagaa agagctgcctcccggggtgg aggagctgct gaacaagggg caggaccctc tggcagacag gggtgagaaggacacagcta agagcctcca gcccttggcg ccccggaaca agacccgtgt ggtcagctaccgcgtgcccc acaacgctgc ggtgcaggtg tacgactacc gagagaagcg agcccgcgtggtcttcgggc ctgagctggt gtcgctgggt cctgaggagc agttcacagt gttgtccctctcagctgggc ggcccaagcg tccccatgcc cgccgtgcgc tctgcctgct gctggggcctgacttcttca cagacgtcat caccatcgaa acggcggatc atgccaggct gcaactgcagctggcctaca actggcactt tgaggtgaat gaccggaagg acccccaaga gacggccaagctcttttcag tgccagactt tgtaggtgat gcctgcaaag ccatcgcatc ccgggtgcggggggccgtgg cctctgtcac tttcgatgac ttccataaga actcagcccg catcattcgcactgctgtct ttggctttga gacctcggaa gcgaagggcc ccgatggcat ggccctgcccaggccccggg accaggctgt cttcccccaa aacgggctgg tggtcagcag tgtggacgtgcagtcagtgg agcctgtgga tcagaggacc cgggacgccc tgcaacgcag cgtccagctggccatcgaga tcaccaccaa ctcccaggaa gcggcggcca agcatgaggc tcagagactggagcaggaag cccgcggccg gcttgagcgg cagaagatcc tggaccagtc agaagccgagaaagctcgca aggaactttt ggagctggag gctctgagca tggccgtgga gagcaccgggactgccaagg cggaggccga gtcccgtgcg gaggcagccc ggattgaggg agaagggtccgtgctgcagg ccaagctaaa agcacaggcc ttggccattg aaacggaggc tgagctccagagggtccaga aggtccgaga gctggaactg gtctatgccc gggcccagct ggagctggaggtgagcaagg ctcagcagct ggctgaggtg gaggtgaaga agttcaagca gatgacagaggccataggcc ccagcaccat cagggacctt gctgtggctg ggcctgagat gcaggtaaaactgctccagt ccctgggcct gaaatcaacc ctcatcaccg atggctccac tcccatcaacctcttcaaca cagcctttgg gctgctgggg atggggcccg agggtcagcc cctgggcagaagggtggcca gtgggcccag ccctggggag gggatatccc cccagtctgc tcaggcccctcaagctcctg gagacaacca cgtggtgcct gtactgcgct aaRat MVP, Genbank #AAC52161 SEQ ID NO: 18Met Ala Thr Glu Glu Ala Ile Ile Arg Ile Pro Pro Tyr His Tyr IleHis Val Leu Asp Gln Asn Ser Asn Val Ser Arg Val Glu Val Gly ProLys Thr Tyr Ile Arg Gln Asp Asn Glu Arg Val Leu Phe Ala Pro ValArg Met Val Thr Val Pro Pro Arg His Tyr Cys Ile Val Ala Asn ProVal Ser Arg Asp Thr Gln Ser Ser Val Leu Phe Asp Ile Thr Gly GlnVal Arg Leu Arg His Ala Asp Gln Glu Ile Arg Leu Ala Gln Asp ProPhe Pro Leu Tyr Prl Gly Glu Val Leu Glu Lys Asp Ile Thr Pro LeuGln Val Val Leu Pro Asn Thr Ala Leu His Leu Lys Ala Leu Leu AspPhe Glu Asp Lys Asn Gly Asp Lys Val Met Ala Gly Asp Glu Trp LeuPhe Glu Gly Pro Gly Thr Tyr Ile Pro Gln Lys Glu Val Glu Val ValGlu Ile Ile Gln Ala Thr Val Ile Lys Gln Asn Gln Ala Leu Arg LeuArg Ala Arg Lys Glu Cys Phe Asp Arg Glu Gly Lys Gly Arg Val ThrGly Glu Glu Trp Leu Val Arg Ser Val Gly Ala Tyr Leu Pro Ala ValPhe Glu Glu Val Leu Asp Leu Val Asp Ala Val Ile Leu Thr Glu LysThr Ala Leu His Leu Arg Ala Leu Gln Asn Phe Arg Asp Leu Arg GlyVal Leu His Arg Thr Gly Glu Glu Trp Leu Val Thr Val Gln Asp ThrGlu Ala His Val Pro Asp Val Tyr Glu Glu Val Leu Gly Val Val ProIle Thr Thr Leu Gly Pro Arg His Tyr Cys Val Ile Leu Asp Pro MetGly Pro Asp Gly Lys Asn Gln Leu Gly Gln Lys Arg Val Val Lys GlyGlu Lys Ser Phe Phe Leu Gln Pro Gly Glu Arg Leu Glu Arg Gly IleGln Asp Val Tyr Val Leu Ser Glu Gln Gln Gly Leu Leu Leu Lys AlaLeu Gln Pro Leu Glu Glu Gly Glu Ser Glu Glu Lys Val Ser His GlnAla Gly Asp Cys Trp Leu Ile Arg Gly Pro Leu Glu Tyr Val Pro SerAla Lys Val Glu Val Val Glu Glu Arg Gln Ala Ile Pro Leu Asp GlnAsn Glu Gly Ile Tyr Val Gln Asp Val Lys Thr Gly Lys Val Arg AlaVal Ile Gly Ser Thr Tyr Met Leu Thr Gln Asp Glu Val Leu Trp GluLys Glu Leu Pro Ser Gly Val Glu Glu Leu Leu Asn Leu Gly His AspPro Leu Ala Asp Arg Gly Gln Lys Gly Thr Ala Lys Pro Leu Gln ProSer Ala Pro Arg Asn Lys Thr Arg Val Val Ser Tyr Arg Val Pro HisAsn Ala Ala Val Gln Val Tyr Asp Tyr Arg Ala Lys Arg Ala Arg ValVal Phe Gly Pro Glu Leu Val Thr Leu Asp Pro Glu Glu Gln Phe ThrVal Leu Ser Leu Ser Ala Gly Arg Pro Lys Arg Pro His Ala Arg ArgAla Leu Cys Leu Leu Leu Gly Pro Asp Phe Phe Thr Asp Val Ile ThrIle Glu Thr Ala Asp His Ala Arg Leu Gln Leu Gln Leu Ala Tyr AsnTrp His Phe Glu Leu Lys Asn Arg Asn Asp Pro Ala Glu Ala Ala LysLeu Phe Ser Val Pro Asp Phe Val Gly Asp Ala Cys Lys Ala Ile AlaSer Arg Val Arg Gly Ala Val Ala Ser Val Thr Phe Asp Asp Phe HisLys Asn Ser Ala Arg Ile Ile Arg Met Ala Val Phe Gly Phe Glu MetSer Glu Asp Thr Gly Pro Asp Gly Thr Leu Leu Pro Lys Ala Arg AspGln Ala Val Phe Pro Gln Asn Gly Leu Val Val Ser Ser Val Asp ValGln Ser Val Glu Pro Val Asp Gln Arg Thr Arg Asp Ala Leu Gln ArgSer Val Gln Leu Ala Ile Glu Ile Thr Thr Asn Ser Gln Glu Ala AlaAla Lys His Glu Ala Gln Arg Leu Glu Gln Glu Ala Arg Gly Arg LeuGlu Arg Gln Lys Ile Leu Asp Gln Ser Glu Ala Glu Lys Ala Arg LysGlu Leu Leu Glu Leu Glu Ala Met Ser Met Ala Val Glu Ser Thr GlyAsn Ala Lys Ala Glu Ala Glu Ser Arg Ala Glu Ala Ala Arg Ile GluGly Glu Gly Ser Val Leu Gln Ala Lys Leu Lys Ala Gln Ala Leu AlaIle Glu Thr Glu Ala Glu Leu Glu Arg Val Lys Lys Val Arg Glu MetGlu Leu Ile Tyr Ala Arg Ala Gln Leu Glu Leu Glu Val Ser Lys AlaGln Gln Leu Ala Asn Val Glu Ala Lys Lys Phe Lys Glu Met Thr GluAla Leu Gly Pro Gly Thr Ile Arg Asp Leu Ala Val Ala Gly Pro GluMet Gln Val Lys Leu Leu Gln Ser Leu Gly Leu Lys Ser Thr Leu IleThr Asp Gly Ser Ser Pro Ile Asn Leu Phe Ser Thr Ala Phe Gly LeuLeu Gly Leu Gly Ser Asp Gly Gln Pro Pro Ala Gln LysRat MVP cDNA, Genbank #U09870 SEQ ID NO: 19atggcaactg aagaggccat catccgcatc cccccatacc actacatcca tgtgctggaccagaacagta atgtgtcccg tgtggaggtt ggaccaaaga cctacatccg gcaggacaatgagagggtac tgtttgcccc agttcgcatg gtgaccgtcc ccccacgcca ctactgcatagtggccaacc ctgtgtcccg ggacacccag agttctgtgt tatttgacat cacaggacaagtccgactcc ggcacgctga ccaggagatc cgactagccc aggacccctt ccccctgtatccaggggagg tgctggaaaa ggacatcacc ccactgcagg tggttctgcc caacacagcactgcatctta aggcgttgct ggactttgag gataagaatg gagacaaggt catggcaggagacgagtggc tatttgaggg acctggcacc tacatcccac agaaggaagt ggaagtcgtggagatcattc aggccacagt catcaaacag aaccaagcac tgcggctaag ggcccgaaaggagtgctttg accgggaggg caaggggcgc gtgacaggtg aggagtggct ggtccgatccgtgggggctt acctcccagc tgtctttgaa gaggtgctgg atctggtgga tgctgtgatccttacagaaa agactgccct gcacctccgg gctctgcaga acttcaggga ccttcggggagtgctccacc gcaccgggga ggaatggtta gtgacagtgc aggacacaga agcccatgttccagatgtct atgaggaggt gcttggggta gtacccatca ccaccctggg acctcgacactactgtgtca ttcttgaccc aatgggacca gacggcaaga accagctggg acaaaagcgtgttgtcaagg gagagaagtc ctttttcctc cagccaggag agaggctgga gcgaggcatccaggatgtgt atgtgctgtc agagcagcag gggctgctac tgaaggcact gcagcccctggaggagggag agagcgagga gaaggtctcc catcaggccg gagactgctg gctcatccgtgggcccctgg agtatgtgcc atctgcaaaa gtggaggtgg tggaggagcg tcaggctatccctctggacc aaaatgaggg catctatgtg caggatgtca agacggggaa ggtgcgggctgtgattggaa gcacctacat gctgactcag gatgaagtcc tgtgggaaaa ggagctgccttctggggtgg aggagctgct gaacttgggg catgaccctc tggcagacag gggtcagaagggcacagcca agccccttca gccctcagct ccaaggaaca agacccgagt ggtcagctaccgtgtcccgc acaatgcagc ggtgcaggtc tatgactaca gagccaagag agcccgtgtggtctttgggc ccgagctagt gacactggat cctgaggagc agttcacagt attgtccctttctgccgggc gacccaagcg tcctcatgcc cgccgtgcac tctgcctact gctgggacctgatttcttta ctgatgtcat caccatcgaa actgcagatc atgccaggtt gcagctgcagcttgcctaca actggcactt tgaactgaag aaccggaatg accctgcaga ggcagccaagcttttctccg tgcctgactt cgtgggtgac gcctgcaagg ccattgcatc ccgagtccggggggctgtag cctctgtcac ctttgatgac ttccataaaa actcagcccg gatcattcgaatggctgttt ttggctttga gatgtctgaa gacacaggtc ctgatggcac actcctgcccaaggctcgag accaggcagt ctttccccaa aacgggctgg tagtcagcag tgtggatgtgcagtcagtgg agcccgtgga ccagaggacc cgggatgccc ttcagcgcag cgttcagctggccatcgaaa ttaccaccaa ctcccaggag gcagcagcca agcacgaggc tcagagactggaacaggaag cccgtggtcg gcttgagagg cagaagatct tggaccagtc agaagctgaaaaagcccgca aggaactctt ggagcttgag gctatgagca tggctgtgga gagcacgggtaatgccaaag cagaggctga gtcccgtgca gaggcagcga ggatcgaagg agaaggctctgtgctgcagg ccaagctcaa ggcacaggcg ctagccattg agacggaggc tgagttggagcgagtaaaga aagtacgaga gatggaactg atctatgccc gggcccagtt ggagctggaggtgagcaagg cgcagcagct tgccaatgtg gaggcaaaga agttcaagga gatgacagaggcactgggcc ccggcaccat cagggacctg gctgtggccg ggccagagat gcaggtgaaacttctccagt ccctgggcct gaaatccact ctcatcaccg atggctcgtc tcccatcaacctcttcagca cagccttcgg gttgctgggg ctggggtctg atggtcagcc gccagcacag aagtgaCP Peptide SEQ ID NO: 20 Met Ala Gly Cys Gly Cys Pro Cys Gly Cys Gly AlaHuman CP-MVP SEQ ID NO: 21Met Ala Gly Cys Gly Cys Pro Cys Gly Cys Gly Ala Met Ala Thr GluGlu Phe Ile Ile Arg Ile Pro Pro Tyr His Tyr Ile His Val Leu AspGln Asn Ser Asn Val Ser Arg Val Glu Val Gly Pro Lys Thr Tyr IleArg Gln Asp Asn Glu Arg Val Leu Phe Ala Pro Met Arg Met Val ThrVal Pro Pro Arg His Tyr Cys Thr Val Ala Asn Pro Val Ser Arg AspAla Gln Gly Leu Val Leu Phe Asp Val Thr Gly Gln Val Arg Leu ArgHis Ala Asp Leu Glu Ile Arg Leu Ala Gln Asp Pro Phe Pro Leu TyrPro Gly Glu Val Leu Glu Lys Asp Ile Thr Pro Leu Gln Val Val LeuPro Asn Thr Ala Leu His Leu Lys Ala Leu Leu Asp Phe Glu Asp LysAsp Gly Asp Lys Val Val Ala Gly Asp Glu Trp Leu Phe Glu Gly ProGly Thr Tyr Ile Pro Arg Lys Glu Val Glu Val Val Glu Ile Ile GlnAla Thr Ile Ile Arg Gln Asn Gln Ala Leu Arg Leu Arg Ala Arg LysGlu Cys Trp Asp Arg Asp Gly Lys Glu Arg Val Thr Gly Glu Glu TrpLeu Val Thr Thr Val Gly Ala Tyr Leu Pro Ala Val Phe Glu Glu ValLeu Asp Leu Val Asp Ala Val Ile Leu Thr Glu Lys Thr Ala Leu HisLeu Arg Ala Arg Arg Asn Phe Arg Asp Phe Arg Gly Val Ser Arg ArgThr Gly Glu Glu Trp Leu Val Thr Val Gln Asp Thr Glu Ala His ValPro Asp Val His Glu Glu Val Leu Gly Val Val Pro Ile Thr Thr LeuGly Pro His Asn Tyr Cys Val Ile Leu Asp Pro Val Gly Pro Asp GlyLys Asn Gln Leu Gly Gln Lys Arg Val Val Lys Gly Glu Lys Ser PhePhe Leu Gln Pro Gly Glu Gln Leu Glu Gln Gly Ile Gln Asp Val TyrVal Leu Ser Glu Gln Gln Gly Leu Leu Leu Arg Ala Leu Gln Pro LeuGlu Glu Gly Glu Asp Glu Glu Lys Val Ser His Gln Ala Gly Asp HisTrp Leu Ile Arg Gly Pro Leu Glu Tyr Val Pro Ser Ala Lys Val GluVal Val Glu Glu Arg Gln Ala Ile Pro Leu Asp Glu Asn Glu Gly IleTyr Val Gln Asp Val Lys Thr Gly Lys Val Arg Ala Val Ile Gly SerThr Tyr Met Leu Thr Gln Asp Glu Val Leu Trp Glu Lys Glu Leu ProPro Gly Val Glu Glu Leu Leu Asn Lys Gly Gln Asp Pro Leu Ala AspArg Gly Glu Lys Asp Thr Ala Lys Ser Leu Gln Pro Leu Ala Pro ArgAsn Lys Thr Arg Val Val Ser Tyr Arg Val Pro His Asn Ala Ala ValGln Val Tyr Asp Tyr Arg Glu Lys Arg Ala Arg Val Val Phe Gly ProGlu Leu Val Ser Leu Gly Pro Glu Glu Gln Phe Thr Val Leu Ser LeuSer Ala Gly Arg Pro Lys Arg Pro His Ala Arg Arg Ala Leu Cys LeuLeu Leu Gly Pro Asp Phe Phe Thr Asp Val Ile Thr Ile Glu Thr AlaAsp His Ala Arg Leu Gln Leu Gln Leu Ala Tyr Asn Trp His Phe GluVal Asn Asp Arg Lys Asp Pro Gln Glu Thr Ala Lys Leu Phe Ser ValPro Asp Phe Val Gly Asp Ala Cys Lys Ala Ile Ala Ser Arg Val ArgGly Ala Val Ala Ser Val Thr Phe Asp Asp Phe His Lys Asn Ser AlaArg Ile Ile Arg Thr Ala Val Phe Gly Phe Glu Thr Ser Glu Ala LysGly Pro Asp Gly Met Ala Leu Pro Arg Pro Arg Asp Gln Ala Val PhePro Gln Asn Gly Leu Val Val Ser Ser Val Asp Val Gln Ser Val GluPro Val Asp Gln Arg Thr Arg Asp Ala Leu Gln Arg Ser Val Gln LeuAla Ile Glu Ile Thr Thr Asn Ser Gln Glu Ala Ala Ala Lys His GluAla Gln Arg Leu Glu Gln Glu Ala Arg Gly Arg Leu Glu Arg Gln LysIle Leu Asp Gln Ser Glu Ala Glu Lys Ala Arg Lys Glu Leu Leu GluLeu Glu Ala Leu Ser Met Ala Val Glu Ser Thr Gly Thr Ala Lys AlaGlu Ala Glu Ser Arg Ala Glu Ala Ala Arg Ile Glu Gly Glu Gly SerVal Leu Gln Ala Lys Leu Lys Ala Gln Ala Leu Ala Ile Glu Thr GluAla Glu Leu Gln Arg Val Gln Lys Val Arg Glu Leu Glu Leu Val TyrAla Arg Ala Gln Leu Glu Leu Glu Val Ser Lys Ala Gln Gln Leu AlaGlu Val Glu Val Lys Lys Phe Lys Gln Met Thr Glu Ala Ile Gly ProSer Thr Ile Arg Asp Leu Ala Val Ala Gly Pro Glu Met Gln Val LysLeu Leu Gln Ser Leu Gly Leu Lys Ser Thr Leu Ile Thr Asp Gly SerThr Pro Ile Asn Leu Phe Asn Thr Ala Phe Gly Leu Leu Gly Met GlyPro Glu Gly Gln Pro Leu Gly Arg Arg Val Ala Ser Gly Pro Ser ProGly Glu gly Ile Ser Pro Gln Ser Ala Gln Ala Pro Gln Ala Pro GlyAsp Asn His Val Val Pro Val Leu Arg Human CP-MVP cDNA SEQ ID NO: 22atggcaggct gcggttgtcc atgcggttgt ggcgccatgg caactgaaga gttcatcatccgcatccccc cataccacta tatccatgtg ctggaccaga acagcaacgt gtcccgtgtggaggtcgggc caaagaccta catccggcag gacaatgaga gggtactgtt tgcccccatgcgcatggtga ccgtcccccc acgtcactac tgcacagtgg ccaaccctgt gtctcgggatgcccagggct tggtgctgtt tgatgtcaca gggcaagttc ggcttcgcca cgctgacctcgagatccggc tggcccagga ccccttcccc ctgtacccag gggaggtgct ggaaaaggacatcacacccc tgcaggtggt tctggggaac actgccctcc atctaaaggc gctgcttgattttgaggata aagatggaga caaggtggtg gcaggagatg agtggctttt cgagggacctggcacgtaca tcccccggaa ggaagtggag gtcgtggaga tcattcaggc caccatcatcaggcagaacc aggctctgcg gctcagggcc cgcaaggagt gctgggaccg ggacggcaaggagagggtga caggggaaga atggctggtc accacagtag gggcgtacct cccagcggtgtttgaggagg ttctggattt ggtggacgcc gtcatcctta cggaaaagac agccctgcacctccgggctc ggcggaactt ccgggacttc aggggagtgt cccgccgcac tggggaggagtggctggtaa cagtgcagga cacagaggcc cacgtgccag atgtccacga ggaggtgctgggggttgtgc ccatcaccac cctgggcccc cacaactact gcgtgattct cgaccctgtcggaccggatg gcaagaatca gctggggcag aagcgcgtgg tcaagggaga gaagtcttttttcctccagc caggagagca gctggaacaa ggcatccagg atgtgtatgt gctgtcggagcagcaggggc tgctgctgag ggccctgcag cccctggagg agggggagga tgaggagaaggtctcacacc aggctgggga ccactggctc atccgcggac ccctggagta tgtgccatctgccaaagtgg aggtggtgga ggagcgccag gccatccctc tagacgagaa cgagggcatctatgtgcagg atgtcaagac cggaaaggtg cgcgctgtga ttggaagcac ctacatgctgacccaggacg aagtcctgtg ggagaaagag ctgcctcccg gggtggagga gctgctgaacaaggggcagg accctctggc agacaggggt gagaaggaca cagctaagag cctccagcccttggcgcccc ggaacaagac ccgtgtggtc agctaccgcg tgccccacaa cgctgcggtgcaggtgtacg actaccgaga gaagcgagcc cgcgtggtct tcgggcctga gctggtgtcgctgggtcctg aggagcagtt cacagtgttg tccctctcag ctgggcggcc caagcgtccccatgcccgcc gtgcgctctg cctgctgctg gggcctgact tcttcacaga cgtcatcaccatcgaaacgg cggatcatgc caggctgcaa ctgcagctgg cctacaactg gcactttgaggtgaatgacc ggaaggaccc ccaagagacg gccaagctct tttcagtgcc agactttgtaggtgatgcct gcaaagccat cgcatcccgg gtgcgggggg ccgtggcctc tgtcactttcgatgacttcc ataagaactc agcccgcatc attcgcactg ctgtctttgg ctttgagacctcggaagcga agggccccga tggcatggcc ctgcccaggc cccgggacca ggctgtcttcccccaaaacg ggctggtggt cagcagtgtg gacgtgcagt cagtggagcc tgtggatcagaggacccggg acgccctgca acgcagcgtc cagctggcca tcgagatcac caccaactcccaggaagcgg cggccaagca tgaggctcag agactggagc aggaagcccg cggccggcttgagcggcaga agatcctgga ccagtcagaa gccgagaaag ctcgcaagga acttttggagctggaggctc tgagcatggc cgtggagagc accgggactg ccaaggcgga ggccgagtcccgtgcggagg cagcccggat tgagggagaa gggtccgtgc tgcaggccaa gctaaaagcacaggccttgg ccattgaaac ggaggctgag ctccagaggg tccagaaggt ccgagagctggaactggtct atgcccgggc ccagctggag ctggaggtga gcaaggctca gcagctggctgaggtggagg tgaagaagtt caagcagatg acagaggcca taggccccag caccatcagggaccttgctg tggctgggcc tgagatgcag gtaaaactgc tccagtccct gggcctgaaatcaaccctca tcaccgatgg ctccactccc atcaacctct tcaacacagc ctttgggctgctggggatgg ggcccgaggg tcagcccctg ggcagaaggg tggccagtgg gcccagcctcggggagggga tatcccccca gtctgctcag gcccctcaag ctcctggaga caaccacgtggtgcctgtac tgcgctaa Rat CP-MVP SEQ ID NO: 23Met Ala Gly Cys Gly Cys Pro Cys Gly Cys Gly Ala Met Ala Thr GluGlu Ala Ile Ile Arg Ile Pro Pro Tyr His Tyr Ile His Val Leu AspGln Asn Ser Asn Val Ser Arg Val Glu Val Gly Pro Lys Thr Tyr IleArg Gln Asp Asn Glu Arg Val Leu Phe Ala Pro Val Arg Met Val ThrVal Pro Pro Arg His Tyr Cys Ile Val Ala Asn Pro Val Ser Arg AspThr Gln Ser Ser Val Leu Phe Asp Ile Thr Gly Gln Val Arg Leu ArgHis Ala Asp Gln Glu Ile Arg Leu Ala Gln Asp Pro Phe Pro Leu TyrPro Gly Glu Val Leu Glu Lys Asp Ile Thr Pro Leu Gln Val Val LeuPro Asn Thr Ala Leu His Leu Lys Ala Leu Leu Asp Phe Glu Asp LysAsn Gly Asp Lys Val Met Ala Gly Asp Glu Trp Leu Phe Glu Gly ProGly Thr Tyr Ile Pro Gln Lys Glu Val Glu Val Val Glu Ile Ile GlnAla Thr Val Ile Lys Gln Asn Gln Ala Leu Arg Leu Arg Ala Arg LysGlu Cys Phe Asp Arg Glu Gly Lys Gly Arg Val Thr Gly Glu Glu TrpLeu Val Arg Ser Val Gly Ala Tyr Leu Pro Ala Val Phe Glu Glu ValLue Asp Leu Val Asp Ala Val Ile Leu Thr Glu Lys Thr Ala Leu HisLeu Arg Ala Leu Gln Asn Phe Arg Asp Leu Arg Gly Val Leu His ArgThr Gly Glu Glu Trp Leu Val Thr Val Gln Asp Thr Glu Ala His ValPro Asp Val Tyr Glu Glu Val Leu Gly Val Val Pro Ile Thr Thr LeuGly Pro Arg His Tyr Cys Val Ile Leu Asp Pro Met Gly Pro Asp GlyLys Asn Gln Leu Gly Gln Lys Arg Val Val Lys Gly Glu Lys Ser PhePhe Leu Gln Pro Gly Glu Arg Leu Glu Arg Gly Ile Gln Asp Val TyrVal Leu Ser Glu Gln Gln Gly Leu Leu Leu Lys Ala Leu Gln Pro LeuGlu Glu Gly Glu Ser Glu Glu Lys Val Ser His Gln Ala Gly Asp CysTrp Leu Ile Arg Gly Pro Leu Glu Tyr Val Pro Ser Ala Lys Val GluVal Val Glu Glu Arg Gln Ala Ile Pro Leu Asp Gln Asn Glu Gly IleTyr Val Gln Asp Val Lys Thr Gly Lys Val Arg Ala Val Ile Gly SerThr Tyr Met Leu Thr Gln Asp Glu Val Leu Trp Glu Lys Glu Leu ProSer Gly Val Glu Glu Leu Leu Asn Leu Gly His Asp Pro Leu Ala AspArg Gly Gln Lys Gly Thr Ala Lys Pro Leu Gln Pro Ser Ala Pro ArgAsn Lys Thr Arg Val Val Ser Tyr Arg Val Pro His Asn Ala Ala ValGln Val Tyr Asp Tyr Arg Ala Lys Arg Ala Arg Val Val Phe Gly ProGlu Leu Val Thr Leu Asp Pro Glu Glu Gln Phe Thr Val Leu Ser LeuSer Ala Gly Arg Pro Lys Arg Pro His Ala Arg Arg Ala Leu Cys LeuLeu Leu Gly Pro Asp Phe Phe Thr Asp Val Ile Thr Ile Glu Thr AlaAsp His Ala Arg Leu Gln Leu Gln Leu Ala Tyr Asn Trp His Phe GluLeu Lys Asn Arg Asn Asp Pro Ala Glu Ala Ala Lys Leu Phe Ser ValPro Asp Phe Val Gly Asp Ala Cys Lys Ala Ile Ala Ser Arg Val ArgGly Ala Val Ala Ser Val Thr Phe Asp Asp Phe His Lys Asn Ser AlaArg Ile Ile Arg Met Ala Val Phe Gly Phe Glu Met Ser Glu Asp ThrGly Pro Asp Gly Thr Leu Leu Pro Lys Ala Arg Asp Gln Ala Val PhePro Gln Asn Gly Leu Val Val Ser Ser Val Asp Val Gln Ser Val GluPro Val Asp Gln Arg Thr Arg Asp Ala Leu Gln Arg Ser Val Gln LeuAla Ile Glu Ile Thr Thr Asn Ser Gln Glu Ala Ala Ala Lys His GluAla Gln Arg Leu Glu Gln Glu Ala Arg Gly Arg Leu Glu Arg Gln LysIle Leu Asp Gln Ser Glu Ala Glu Lys Ala Arg Lys Glu Leu Leu GluLeu Glu Ala Met Ser Met Ala Val Glu Ser Thr Gly Asn Ala Lys AlaGlu Ala Glu Ser Arg Ala Glu Ala Ala Arg Ile Glu Gly Glu Gly SerVal Leu Gln Ala Lys Leu Lys Ala Gln Ala Leu Ala Ile Glu Thr GluAla Glu Leu Glu Arg Val Lys Lys Val Arg Glu Met Glu Leu Ile TyrAla Arg Ala Gln Leu Glu Leu Glu Val Ser Lys Ala Gln Gln Leu AlaAsn Val Glu Ala Lys Lys Phe Lys Glu Met Thr Glu Ala Leu Gly ProGly Thr Ile Arg Asp Leu Ala Val Ala Gly Pro Glu Met Gln Val LysLeu Leu Gln Ser Leu Gly Leu Lys Ser Thr Leu Ile Thr Asp Gly SerSer Pro Ile Asn Leu Phe Ser Thr Ala Phe Gly Leu Leu Gly Leu GlySer Asp Gly Gln Pro Pro Ala Gln Lys Rat CP-MVP cDNA SEQ ID NO: 24atggcaggct gcggttgtcc atgcggttgt ggcgccatgg caactgaaga ggccatcatccgcatccccc cataccacta catccatgtg ctggaccaga acagtaatgt gtcccgtgtggaggttggac caaagaccta catccggcag gacaatgaga gggtactgtt tgccccagttcgcatggtga ccgtcccccc acgccactac tgcatagtgg ccaaccctgt gtcccgggacacccagagtt ctgtgttatt tgacatcaca ggacaagtcc gactccggca cgctgaccaggagatccgac tagcccagga ccccttcccc ctgtatccag gggaggtgct ggaaaaggacatcaccccac tgcaggtggt tctgcccaac acagcactgc atcttaaggc gttgctggactttgaggata agaatggaga caaggtcatg gcaggagacg agtggctatt tgagggacctggcacctaca tcccacagaa ggaagtggaa gtcgtggaga tcattcaggc cacagtcatcaaacagaacc aagcactgcg gctaagggcc cgaaaggagt gctttgaccg ggagggcaaggggcgcgtga caggtgagga gtggctggtc cgatccgtgg gggcttacct cccagctgtctttgaagagg tgctggatct ggtggatgct gtgatcctta cagaaaagac tgccctgcacctccgggctc tgcagaactt cagggacctt cggggagtgc tccaccgcac cggggaggaatggttagtga cagtgcagga cacagaagcc catgttccag atgtctatga ggaggtgcttggggtagtac ccatcaccac cctgggacct cgacactact gtgtcattct tgacccaatgggaccagacg gcaagaacca gctgggacaa aagcgtgttg tcaagggaga gaagtcctttttcctccagc caggagagag gctggagcga ggcatccagg atgtgtatgt gctgtcagagcagcaggggc tgctactgaa ggcactgcag cccctggagg agggagagag cgaggagaaggtctcccatc aggccggaga ctgctggctc atccgtgggc ccctggagta tgtgccatctgcaaaagtgg aggtggtgga ggagcgtcag gctatccctc tggaccaaaa tgagggcatctatgtgcagg atgtcaagac ggggaaggtg cgggctgtga ttggaagcac ctacatgctgactcaggatg aagtcctgtg ggaaaaggag ctgccttctg gggtggagga gctgctgaacttggggcatg accctctggc agacaggggt cagaagggca cagccaagcc ccttcagccctcagctccaa ggaacaagac ccgagtggtc agctaccgtg tcccgcacaa tgcagcggtgcaggtctatg actacagagc caagagagcc cgtgtggtct ttgggcccga gctagtgacactggatcctg aggagcagtt cacagtattg tccctttctg ccgggcgacc caagcgtcctcatgcccgcc gtgcactctg cctactgctg ggacctgatt tctttactga tgtcatcaccatcgaaactg cagatcatgc caggttgcag ctgcagcttg cctacaactg gcactttgaactgaagaacc ggaatgaccc tgcagaggca gccaagcttt tctccgtgcc tgacttcgtgggtgacgcct gcaaggccat tgcatcccga gtccgggggg ctgtagcctc tgtcacctttgatgacttcc ataaaaactc agcccggatc attcgaatgg ctgtttttgg ctttgagatgtctgaagaca caggtcctga tggcacactc ctgcccaagg ctcgagacca ggcagtctttccccaaaacg ggctggtagt cagcagtgtg gatgtgcagt cagtggagcc cgtggaccagaggacccggg atgcccttca gcgcagcgtt cagctggcca tcgaaattac caccaactcccaggaggcag cagccaagca cgaggctcag agactggaac aggaagcccg tggtcggcttgagaggcaga agatcttgga ccagtcagaa gctgaaaaag cccgcaagga actcttggagcttgaggcta tgagcatggc tgtggagagc acgggtaatg ccaaagcaga ggctgagtcccgtgcagagg cagcgaggat cgaaggagaa ggctctgtgc tgcaggccaa gctcaaggcacaggcgctag ccattgagac ggaggctgag ttggagcgag taaagaaagt acgagagatggaactgatct atgcccgggc ccagttggag ctggaggtga gcaaggcgca gcagcttgccaatgtggagg caaagaagtt caaggagatg acagaggcac tgggccccgg caccatcagggacctggctg tggccgggcc agagatgcag gtgaaacttc tccagtccct gggcctgaaatccactctca tcaccgatgg ctcgtctccc atcaacctct tcagcacagc cttcgggttgctggggctgg ggtctgatgg tcagccgcca gcacagaagt gaHuman TEP1, Genbank #AAC51107 SEQ ID NO: 25 Met Glu Lys Leu His Gly His Val Ser Ala His Pro Asp Ile Leu SerLeu Glu Asn Arg Cys Leu Ala Met Leu Pro Asp Leu Gln Pro Leu GluLys Leu His Gln His Val Ser Thr His Ser Asp Ile Leu Ser Leu LysAsn Gln Cys Leu Ala Thr Leu Pro Asp Leu Lys Thr Met Glu Lys ProHis Gly Tyr Val Ser Ala His Pro Asp Ile Leu Ser Leu Glu Asn GlnCys Leu Ala Thr Leu Ser Asp Leu Lys Thr Met Glu Lys Pro His GlyHis Val Ser Ala His Pro Asp Ile Leu Ser Leu Glu Asn Arg Cys LeuAla Thr Leu Pro Ser Leu Lys Ser Thr Val Ser Ala Ser Pro Leu PheGln Ser Leu Gln Ile Ser His Met Thr Gln Ala Asp Leu Tyr Arg ValAsn Asn Ser Asn Cys Leu Leu Ser Glu Pro Pro Ser Trp Arg Ala GlnHis Phe Ser Lys Gly Leu Asp Leu Ser Thr Cys Pro Ile Ala Leu LysSer Ile Ser Ala Thr Glu Thr Ala Gln Glu Ala Thr Leu Gly Arg TrpPhe Asp Ser Glu Glu Lys Lys Gly Ala Glu Thr Gln Met Pro Ser TyrSer Leu Ser Leu Gly Glu Glu Glu Glu Val Glu Asp Leu Ala Val LysLeu Thr Ser Gly Asp Ser Glu Ser His Pro Glu Pro Thr Asp His ValLeu Gln Glu Lys Lys Met Ala Leu Leu Ser Leu Leu Cys Ser Thr LeuVal Ser Glu Val Asn Met Asn Asn Thr Ser Asp Pro Thr Leu Ala AlaIle Phe Glu Ile Cys Arg Glu Leu Ala Leu Leu Glu Pro Glu Phe IleLeu Lys Ala Ser Leu Tyr Ala Arg Gln Gln Leu Asn Val Arg Asn ValAla Asn Asn Ile Leu Ala Ile Ala Ala Phe Leu Pro Ala Cys Arg ProHis Leu Arg Arg Tyr Phe Cys Ala Ile Val Gln Leu Pro Ser Asp TrpIle Gln Val Ala Glu Leu Tyr Gln Ser Leu Ala Glu Gly Asp Lys AsnLys Leu Val Pro Leu Pro Ala Cys Leu Arg Thr Ala Met Thr Asp LysPhe Ala Gln Phe Asp Glu Tyr Gln Leu Ala Lys Tyr Asn Pro Arg LysHis Arg Ala Lys Arg His Pro Arg Arg Pro Pro Arg Ser Pro Gly MetGlu Pro Pro Phe Ser His Arg Cys Phe Pro Arg Tyr Ile Gly Phe LeuArg Glu Glu Gln Arg Lys Phe Glu Lys Ala Gly Asp Thr Val Ser GluLys Lys Asn Pro Pro Arg Phe Thr Leu Lys Lys Leu Val Gln Arg LeuHis Ile His Lys Pro Ala Gln His Val Gln Ala Leu Leu Gly Tyr ArgTyr Pro Ser Asn Leu Gln Leu Phe Ser Arg Ser Arg Leu Pro Gly ProTrp Asp Ser Ser Arg Ala Gly Lys Arg Met Lys Leu Ser Arg Pro GluThr Trp Glu Arg Glu Leu Ser Leu Arg Gly Asn Lys Ala Ser Val TrpGlu Glu Leu Ile Glu Asn Gly Lys Leu Pro Phe Met Ala Met Leu ArgAsn Leu Cys Asn Leu Leu Arg Val Gly Ile Ser Ser Arg His His GluLeu Ile Leu Gln Arg Leu Gln His Gly Lys Ser Val Ile His Ser ArgGln Phe Pro Phe Arg Phe Leu Asn Ala His Asp Ala Ile Asp Ala LeuGlu Ala Gln Leu Arg Asn Gln Ala Leu Pro Phe Pro Ser Asn Ile ThrLeu Met Arg Arg Ile Leu Thr Arg Asn Glu Lys Asn Arg Pro Arg ArgArg Phe Leu Cys His Leu Ser Arg Gln Gln Leu Arg Met Ala Met ArgIle Pro Val Leu Tyr Glu Gln Leu Lys Arg Glu Lys Leu Arg Val HisLys Ala Arg Gln Trp Lys Tyr Asp Gly Glu Met Leu Asn Arg Tyr ArgGln Ala Leu Glu Thr Ala Val Asn Leu Ser Val Lys His Ser Leu ProLeu Leu Pro Gly Arg Thr Val Leu Val Tyr Leu Thr Asp Ala Asn AlaAsp Arg Leu Cys Pro Lys Ser Asn Pro Gln Gly Pro Pro Leu Asn TyrAla Leu Leu Leu Ile Gly Met Met Ile Thr Arg Ala Glu Gln Val AspVal Val Leu Cys Gly Gly Asp Thr Leu Lys Thr Ala Val Leu Lys AlaGlu Glu Gly Ile Leu Lys Thr Ala Ile Lys Leu Gln Ala Gln Val GlnGlu Phe Asp Glu Asn Asp Gly Trp Ser Leu Asn Thr Phe Gly Lys TyrLeu Leu Ser Leu Ala Gly Gln Arg Val Pro Val Asp Arg Val Ile LeuLeu Gly Gln Ser Met Asp Asp Gly Met Ile Asn Val Ala Lys Gln LeuTyr Trp Gln Arg Val Asn Ser Lys Cys Leu Phe Val Gly Ile Leu LeuArg Arg Val Gln Tyr Leu Ser Thr Asp Leu Asn Pro Asn Asp Val ThrLeu Ser Gly Cys Thr Asp Ala Ile Leu Lys Phe Ile Ala Glu His GlyAla Ser His Leu Leu Glu His Val Gly Gln Met Asp Lys Ile Phe LysIle Pro Pro Pro Pro Gly Lys Thr Gly Val Gln Ser Leu Arg Pro LeuGlu Glu Asp Thr Pro Ser Pro Leu Ala Pro Val Ser Gln Gln Gly TrpArg Ser Ile Arg Leu Phe Ile Ser Ser Thr Phe Arg Asp Met His GlyGlu Arg Asp Leu Leu Leu Arg Ser Val Leu Pro Ala Leu Gln Ala ArgAla Ala Pro His Arg Ile Ser Leu His Gly Ile Asp Leu Arg Trp GlyVal Thr Glu Glu Glu Thr Arg Arg Asn Arg Gln Leu Glu Val Cys LeuGly Glu Val Glu Asn Ala Gln Leu Phe Val Gly Ile Leu Gly Ser ArgTyr Gly Tyr Ile Pro Pro Ser Tyr Asn Leu Pro Asp His Pro His PheHis Trp Ala Gln Gln Tyr Pro Ser Gly Arg Ser Val Thr Glu Met GluVal Met Gln Phe Leu Asn Arg Asn Gln Arg Leu Gln Pro Ser AlaGln Ala Leu Ile Tyr Phe Arg Asp Ser Ser Phe Leu Ser Ser ValPro Asp Ala Trp Lys Ser Asp Phe Val Ser Glu Ser Glu Glu AlaAla Cys Arg Ile Ser Glu Leu Lys Ser Tyr Leu Ser Arg Gln LysGly Ile Thr Cys Arg Arg Tyr Pro Cys Glu Trp Gly Gly Val AlaAla Gly Arg Pro Tyr Val Gly Gly Leu Glu Glu Phe Gly Gln LeuVal Leu Gln Asp Val Trp Asn Met Ile Gln Lys Leu Tyr Leu GlnPro Gly Ala Leu Leu Glu Gln Pro Val Ser Ile Pro Asp Asp AspLeu Val Gln Ala Thr Phe Gln Gln Leu Gln Lys Pro Pro Ser ProAla Arg Pro Arg Leu Leu Gln Asp Thr Val Gln Gln Leu Met LeuPro His Gly Arg Leu Ser Leu Val Thr Gly Gln Ser Gly Gln GlyLys Thr Ala Phe Leu Ala Ser Leu Val Ser Ala Leu Gln Ala ProAsp Gly Ala Lys Val Ala Pro Leu Val Phe Phe His Phe Ser GlyAla Arg Pro Asp Gln Gly Leu Ala Leu Thr Leu Leu Arg Arg LeuCys Thr Tyr Leu Arg Gly Gln Leu Lys Glu Pro Gly Ala Leu ProSer Thr Tyr Arg Ser Leu Val Trp Glu Leu Gln Gln Arg Leu LeuPro Lys Ser Ala Glu Ser Leu His Pro Gly Gln Thr Gln Val LeuIle Ile Asp Gly Ala Asp Arg Leu Val Asp Gln Asn Gly Gln LeuIle Ser Asp Trp Ile Pro Lys Lys Leu Pro Arg Cys Val His LeuVal Leu Ser Val Ser Ser Asp Ala Gly Leu Gly Glu Thr Leu GluGln Ser Gln Gly Ala His Val Leu Ala Leu Gly Pro Leu Glu AlaSer Ala Arg Ala Arg Leu Val Arg Glu Glu Leu Ala Leu Tyr GlyLys Arg Leu Glu Glu Ser Pro Phe Asn Asn Gln Met Arg Leu LeuLeu Val Lys Arg Glu Ser Gly Arg Pro Leu Tyr Leu Arg Leu ValThr Asp His Leu Arg Leu Phe Thr Leu Tyr Glu Gln Val Ser GluArg Leu Arg Thr Leu Pro Ala Thr Val Pro Leu Leu Leu Gln HisIle Leu Ser Thr Leu Glu Lys Glu His Gly Pro Asp Val Leu ProGln Ala Leu Thr Ala Leu Glu Val Thr Arg Ser Gly Leu Thr ValAsp Gln Leu His Gly Val Leu Ser Val Trp Arg Thr Leu Pro LysGly Thr Lys Ser Trp Glu Glu Ala Val Ala Ala Gly Asn Ser GlyAsp Pro Tyr Pro Met Gly Pro Phe Ala Cys Leu Val Gln Ser LeuArg Ser Leu Leu Gly Glu Gly Pro Leu Glu Arg Pro Gly Ala ArgLeu Cys Leu Pro Asp Gly Pro Leu Arg Thr Ala Ala Lys Arg CysTyr Gly Lys Arg Pro Gly Leu Glu Asp Thr Ala His Ile Leu IleAla Ala Gln Leu Trp Lys Thr Cys Asp Ala Asp Ala Ser Gly ThrPhe Arg Ser Cys Pro Pro Glu Ala Leu Gly Asp Leu Pro Tyr HisLeu Leu Gln Ser Gly Asn Arg Gly Leu Leu Ser Lys Phe Leu ThrAsn Leu His Val Val Ala Ala His Leu Glu Leu Gly Leu Val SerArg Leu Leu Glu Ala His Ala Leu Tyr Ala Ser Ser Val Pro LysGlu Glu Gln Lys Leu Pro Glu Ala Asp Val Ala Val Phe Arg ThrPhe Leu Arg Gln Gln Ala Ser Ile Leu Ser Gln Tyr Pro Arg LeuLeu Pro Gln Gln Ala Ala Asn Gln Pro Leu Asp Ser Pro Leu CysHis Gln Ala Ser Leu Leu Ser Arg Arg Trp His Leu Gln His ThrLeu Arg Trp Leu Asn Lys Pro Arg Thr Met Lys Asn Gln Gln SerSer Ser Leu Ser Leu Ala Val Ser Ser Ser Pro Thr Ala Val AlaPhe Ser Thr Asn Gly Gln Arg Ala Ala Val Gly Thr Ala Asn GlyThr Val Tyr Leu Leu Asp Leu Arg Thr Trp Gln Glu Glu Lys SerVal Val Ser Gly Cys Asp Gly Ile Ser Ala Cys Leu Phe Leu SerAsp Asp Thr Leu Phe Leu Thr Ala Phe Asp Gly Leu Leu Glu LeuTrp Asp Leu Gln His Gly Cys Arg Val Leu Gln Thr Lys Ala HisGln Tyr Gln Ile Thr Gly Cys Cys Leu Ser Pro Asp Cys Arg LeuLeu Ala Thr Val Cys Leu Gly Gly Cys Leu Lys Leu Trp Asp ThrVal Arg Gly Gln Leu Ala Phe Gln His Thr Tyr Pro Lys Ser LeuAsn Cys Val Ala Phe His Pro Glu Gly Gln Val Ile Ala Thr GlySer Trp Ala Gly Ser Ile Ser Phe Phe Gln Val Asp Gly Leu LysVal Thr Lys Asp Leu Gly Ala Pro Gly Ala Ser Ile Arg Thr LeuAla Phe Asn Val Pro Gly Gly Val Val Ala Val Gly Arg Leu AspSer Met Val Glu Leu Trp Ala Trp Arg Glu Gly Ala Arg Leu AlaAla Phe Pro Ala His His Gly Phe Val Ala Ala Ala Leu Phe LeuHis Ala Gly Cys Gln Leu Leu Thr Ala Gly Glu Asp Gly Lys ValGln Val Trp Ser Gly Ser Leu Gly Arg Pro Arg Gly His Leu GlySer Leu Ser Leu Ser Pro Ala Leu Ser Val Ala Leu Ser Pro AspGly Asp Arg Val Ala Val Gly Tyr Arg Ala Asp Gly Ile Arg IleTyr Lys Ile Ser Ser Gly Ser Gln Gly Ala Gln Gly Gln Ala LeuAsp Val Ala Val Ser Ala Leu Ala Trp Leu Ser Pro Lys Val LeuVal Ser Gly Ala Glu Asp Gly Ser Leu Gln Gly Trp Ala Leu LysGlu Cys Ser Leu Gln Ser Leu Trp Leu Leu Ser Arg Phe Gln LysPro Val Leu Gly Leu Ala Thr Ser Gln Glu Leu Leu Ala Ser AlaSer Glu Asp Phe Thr Val Gln Leu Trp Pro Arg Gln Leu Leu ThrArg Pro His Lys Ala Glu Asp Phe Pro Cys Gly Thr Glu Leu ArgGly His Glu Gly Pro Val Ser Cys Cys Ser Phe Ser Thr Asp GlyGly Ser Leu Ala Thr Gly Gly Arg Asp Arg Ser Leu Leu Cys TrpAsp Val Arg Thr Pro Lys Thr Pro Val Leu Ile His Ser Phe ProAla Cys His Arg Asp Trp Val Thr Gly Cys Ala Trp Thr Lys AspAsn Leu Leu Ile Ser Cys Ser Ser Asp Gly Ser Val Gly Leu TrpAsp Pro Glu Ser Gly Gln Arg Leu Gly Gln Phe Leu Gly His GlnSer Ala Val Ser Ala Val Ala Ala Val Glu Glu His Val Val SerVal Ser Arg Asp Gly Thr Leu Lys Val Trp Asp His Gln Gly ValGlu Leu Thr Ser Ile Pro Ala His Ser Gly Pro Ile Ser His CysAla Ala Ala Met Glu Pro Arg Ala Ala Gly Gln Pro Gly Ser GluLeu Leu Val Val Thr Val Gly Leu Asp Gly Ala Thr Arg Leu TrpHis Pro Leu Leu Val Cys Gln Thr His Thr Leu Leu Gly His SerGly Pro Val Arg Ala Ala Ala Val Ser Glu Thr Ser Gly Leu MetLeu Thr Ala Ser Glu Asp Gly Ser Val Arg Leu Trp Gln Val ProLys Glu Ala Asp Asp Thr Cys Ile Pro Arg Ser Ser Ala Ala ValThr Ala Val Ala Trp Ala Pro Asp Gly Ser Met Ala Val Ser GlyAsn Gln Ala Gly Glu Leu Ile Leu Trp Gln Glu Ala Lys Ala ValAla Thr Ala Gln Ala Pro Gly His Ile Gly Ala Leu Ile Trp SerSer Ala His Thr Phe Phe Val Leu Ser Ala Asp Glu Lys Ile SerGlu Trp Gln Val Lys Leu Arg Lys Gly Ser Ala Pro Gly Asn LeuSer Leu His Leu Asn Arg Ile Leu Gln Glu Asp Leu Gly Val LeuThr Ser Leu Asp Trp Ala Pro Asp Gly His Phe Leu Ile Leu AlaLys Ala Asp Leu Lys Leu Leu Cys Met Lys Pro Gly Asp Ala ProSer Glu Ile Trp Ser Ser Tyr Thr Glu Asn Pro Met Ile Leu SerThr His Lys Glu Tyr Gly Ile Phe Val Leu Gln Pro Lys Asp ProGly Val Leu Ser Phe Leu Arg Gln Lys Glu Ser Gly Glu Phe GluGlu Arg Leu Asn Phe Asp Ile Asn Leu Glu Asn Pro Ser Arg ThrLeu Ile Ser Ile Thr Gln Ala Lys Pro Glu Ser Glu Ser Ser PheLeu Cys Ala Ser Ser Asp Gly Ile Leu Trp Asn Leu Ala Lys CysSer Pro Glu Gly Glu Trp Thr Thr Gly Asn Met Trp Gln Lys LysAla Asn Thr Pro Glu Thr Gln Thr Pro Gly Thr Asp Pro Ser ThrCys Arg Glu Ser Asp Ala Ser Met Asp Ser Asp Ala Ser Met AspSer Glu Pro Thr Pro His Leu Lys Thr Arg Gln Arg Arg Lys IleHis Ser Gly Ser Val Thr Ala Leu His Val Leu Pro Glu Leu LeuVal Thr Ala Ser Lys Asp Arg Asp Val Lys Leu Trp Glu Arg ProSer Met Gln Leu Leu Gly Leu Phe Arg Cys Glu Gly Ser Val SerCys Leu Glu Pro Trp Leu Gly Ala Asn Ser Thr Leu Gln Leu AlaVal Gly Asp Val Gln Gly Asn Val Tyr Phe Leu Asn Trp GluHuman TEP1 cDNA, Genbank #U86136 SEQ ID NO: 26atggaaaaac tccatgggca tgtgtctgcc catccagaca tcctctcctt ggagaaccggtgcctggcta tgctccctga cttacagccc ttggagaaac tacatcagca tgtatctacccactcagata tcctctcctt gaagaaccag tgcctagcca cgcttcctga cctgaagaccatggaaaaac cacatggata tgtgtctgcc cacccagaca tcctctcctt ggagaaccagtgcctggcca cactttctga cctgaagacc atggagaaac cacatggaca tgtttctgcccacccagaca tcctctcctt ggagaaccgg tgcctggcca ccctccctag tctaaagagcactgtgtctg ccagcccctt gttccagagt ctacagatat ctcacatgac gcaagctgatttgtaccgtg tgaacaacag caattgcctg ctctctgagc ctccaagttg gagggctcagcatttctcta agggactaga cctttcaacc tgccctatag ccctgaaatc catctctgccacagagacag ctcaggaagc aactttgggt cgttggtttg attcagaaga gaagaaaggggcagagaccc aaatgccttc ttatagtctg agcttgggag aggaggagga ggtggaggatctggccgtga agctcacctc tggagactct gaatctcatc cagagcctac tgaccatgtccttcaggaaa agaagatggc tctactgagc ttgctgtgct ctactctggt ctcagaagtaaacatgaaca atacatctga ccccaccctg gctgccattt ttgaaatctg tcgtgaacttgccctcctgg agcctgagtt tatcctcaag gcatctttgt atgccaggca gcagctgaacgtccggaatg tggccaataa catcttggcc attgctgctt tcttgccggc gtgtcgcccccacctgcgac gatatttctg tgccattgtc cagctgcctt ctgactggat ccaggtggctgagctttacc agagcctggc tgagggagat aagaataagc tggtgcccct gcccgcctgtctccgtactg ccatgacgga caaatttgcc cagtttgacg agtaccagct ggctaagtacaaccctcgga agcaccgggc caagagacac ccccgccggc caccccgctc tccagggatggagcctccat tttctcacag atgttttcca aggtacatag ggtttctcag agaagagcagagaaagtttg agaaggccgg tgatacagtg tcagagaaaa agaatcctcc aaggttcaccctgaagaagc tggttcagcg actgcacatc cacaagcctg cccagcacgt tcaagccctgctgggttaca gatacccctc caacctacag ctcttttctc gaagtcgcct tcctgggccttgggattcta gcagagctgg gaagaggatg aagctgtcta ggccagagac ctgggagcgggagctgagcc tacgggggaa caaagcgtcg gtctgggagg aactcattga aaatgggaagcttcccttca tggccatgct tcggaacctg tgcaacctgc tgcgggttgg aatcagttcccgccaccatg agctcattct ccagagactc cagcatggga agtcggtgat ccacagtcggcagtttccat tcagatttct taacgcccat gatgccattg atgccctcga ggctcaactcagaaatcaag cattgccctt tccttcgaat ataacactga tgaggcggat actaactagaaatgaaaaga accgtcccag gcggaggttt ctttgccacc taagccgtca gcagcttcgtatggcaatga ggatacctgt gttgtatgag cagctcaaga gggagaagct gagagtacacaaggccagac agtggaaata tgatggtgag atgctgaaca ggtaccgaca ggccctagagacagctgtga acctctctgt gaagcacagc ctgcccctgc tgccaggccg cactgtcttggtctatctga cagatgctaa tgcagacagg ctctgtccaa agagcaaccc acaagggcccccgctgaact atgcactgct gttgattggg atgatgatca cgagggcgga gcaggtggacgtcgtgctgt gtggaggtga cactctgaag actgcagtgc ttaaggcaga agaaggcatcctgaagactg ccatcaagct ccaggctcaa gtccaggagt ttgatgaaaa tgatggatggtccctgaata cttttgggaa atacctgctg tctctggctg gccaaagggt tcctgtggacagggtcatcc tccttggcca aagcatggat gatggaatga taaatgtggc caaacagctttactggcagc gtgtgaattc caagtgcctc tttgttggta tcctcctaag aagggtacaatacctgtcaa cagatttgaa tcccaatgat gtgacactct caggctgtac tgatgcgatactgaagttca ttgcagagca tggggcctcc catcttctgg aacatgtggg ccaaatggacaaaatattca agattccacc acccccagga aagacagggg tccagtctct ccggccactggaagaggaca ctccaagccc cttggctcct gtttcccagc aaggatggcg cagcatccggcttttcattt catccacttt ccgagacatg cacggggagc gggacctgct gctgaggtctgtgctgccag cactgcaggc ccgagcggcc cctcaccgta tcagccttca cggaatcgacctccgctggg gcgtcactga ggaggagacc cgtaggaaca gacaactgga agtgtgccttggggaggtgg agaacgcaca gctgtttgtg gggattctgg gctcccgtta tggatacattccccccagct acaaccttcc tgaccatcca cacttccact gggcccagca gtacccttcagggcgctctg tgacagagat ggaggtgatg cagttcctga accggaacca acgtctgcagccctctgccc aagctctcat ctacttccgg gattccagct tcctcagctc tgtgccagatgcctggaaat ctgactttgt ttctgagtct gaagaggccg catgtcggat ctcagaactgaagagctacc taagcagaca gaaagggata acctgccgca gatacccctg tgagtgggggggtgtggcag ctggccggcc ctatgttggc gggctggagg agtttgggca gttggttctgcaggatgtat ggaatatgat ccagaagctc tacctgcagc ctggggccct gctggagcagccagtgtcca tcccagacga tgacttggtc caggccacct tccagcagct gcagaagccaccgagtcctg cccggccacg ccttcttcag gacacagtgc aacagctgat gctgccccacggaaggctga gcctggtgac ggggcagtca ggacagggca agacagcctt cctggcatctcttgtgtcag ccctgcaggc tcctgatggg gccaaggtgg caccattagt cttcttccacttttctgggg ctcgtcctga ccagggtctt gccctcactc tgctcagacg cctctgtacctatctgcgtg gccaactaaa agagccaggt gccctcccca gcacctaccg aagcctggtgtgggagctgc agcagaggct gctgcccaag tctgctgagt ccctgcatcc tggccagacccaggtcctga tcatcgatgg ggctgatagg ttagtggacc agaatgggca gctgatttcagactggatcc caaagaagct tccccggtgt gtacacctgg tgctgagtgt gtctagtgatgcaggcctag gggagaccct tgagcagagc cagggtgccc acgtgctggc cttggggcctctggaggcct ctgctcgggc ccggctggtg agagaggagc tggccctgta cgggaagcggctggaggagt caccatttaa caaccagatg cgactgctgc tggtgaagcg ggaatcaggccggccgctct acctgcgctt ggtcaccgat cacctgaggc tcttcacgct gtatgagcaggtgtctgaga gactccggac cctgcctgcc actgtccccc tgctgctgca gcacatcctgagcacactgg agaaggagca cgggcctgat gtccttcccc aggccttgac tgccctagaagtcacacgga gtggtttgac tgtggaccag ctgcacggag tgctgagtgt gtggcggacactaccgaagg ggactaagag ctgggaagaa gcagtggctg ctggtaacag tggagacccctaccccatgg gcccgtttgc ctgcctcgtc cagagtctgc gcagtttgct aggggagggccctctggagc gccctggtgc ccggctgtgc ctccctgatg ggcccctgag aacagcagctaaacgttgct atgggaagag gccagggcta gaggacacgg cacacatcct cattgcagctcagctctgga agacatgtga cgctgatgcc tcaggcacct tccgaagttg ccctcctgaggctctgggag acctgcctta ccacctgctc cagagcggga accgtggact tctttcgaagttccttacca acctccatgt ggtggctgca cacttggaat tgggtctggt ctctcggctcttggaggccc atgccctcta tgcttcttca gtccccaaag aggaacaaaa gctccccgaggctgacgttg cagtgtttcg caccttcctg aggcagcagg cttcaatcct cagccagtacccccggctcc tgccccagca ggcagccaac cagcccctgg actcacctct ttgccaccaagcctcgctgc tctcccggag atggcacctc caacacacac tacgatggct taataaaccccggaccatga aaaatcagca aagctccagc ctgtctctgg cagtttcctc atcccctactgctgtggcct tctccaccaa tgggcaaaga gcagctgtgg gcactgccaa tgggacagtttacctgttgg acctgagaac ttggcaggag gagaagtctg tggtgagtgg ctgtgatggaatctctgctt gtttgttcct ctccgatgat acactctttc ttactgcctt cgacgggctcctggagctct gggacctgca gcatggttgt cgggtgctgc agactaaggc tcaccagtaccaaatcactg gctgctgcct gagcccagac tgccggctgc tagccaccgt gtgcttgggaggatgcctaa agctgtggga cacagtccgt gggcagctgg ccttccagca cacctaccccaagtccctga actgtgttgc cttccaccca gaggggcagg taatagccac aggcagctgggctggcagca tcagcttctt ccaggtggat gggctcaaag tcaccaagga cctgggggcacccggagcct ctatccgtac cttggccttc aatgtgcctg ggggggttgt ggctgtgggccggctggaca gtatggtgga gctgtgggcc tggcgagaag gggcacggct ggctgccttccctgcccacc atggctttgt tgctgctgcg cttttcctgc atgcgggttg ccagttactgacggctggag aggatggcaa ggttcaggtg tggtcagggt ctctgggtcg gccccgtgggcacctgggtt ccctttctct ctctcctgcc ctctctgtgg cactcagccc agatggtgatcgggtggctg ttggatatcg agcggatggc attaggatct acaaaatctc ttcaggttcccagggggctc agggtcaggc actggatgtg gcagtgtccg ccctggcctg gctaagccccaaggtattgg tgagtggtgc agaagatggg tccttgcagg gctgggcact caaggaatgctcccttcagt ccctctggct cctgtccaga ttccagaagc ctgtgctagg actggccacttcccaggagc tcttggcttc tgcctcagag gatttcacag tgcagctgtg gccaaggcagctgctgacgc ggccacacaa ggcagaagac tttccctgtg gcactgagct gcggggacatgagggccctg tgagctgctg tagtttcagc actgatggag gcagcctggc caccgggggccgggatcgga gtctcctctg ctgggacgtg aggacaccca aaacccctgt tttgatccactccttccctg cctgtcaccg tgactgggtc actggctgtg cctggaccaa agataacctactgatatcct gctccagtga tggctctgtg gggctctggg acccagagtc aggacagcggcttggtcagt tcctgggtca tcagagtgct gtgagcgctg tggcagctgt ggaggagcacgtggtgtctg tgagccggga tgggaccttg aaagtgtggg accatcaagg cgtggagctgaccagcatcc ctgctcactc aggacccatt agccactgtg cagctgccat ggagccccgtgcagctggac agcctgggtc agagcttctg gtggtaaccg tcgggctaga tggggccacacggttatggc atccactctt ggtgtgccaa acccacaccc tcctgggaca cagcggcccagtccgtgctg ctgctgtttc agaaacctca ggcctcatgc tgaccgcctc tgaggatggttctgtacggc tctggcaggt tcctaaggaa gcagatgaca catgtatacc aaggagttctgcagccgtca ctgctgtggc ttgggcacca gatggttcca tggcagtatc tggaaatcaagctggggaac taatcttgtg gcaggaagct aaggctgtgg ccacagcaca ggctccaggccacattggtg ctctgatctg gtcctcggca cacacctttt ttgtcctcag tgctgatgagaaaatcagcg agtggcaagt gaaactgcgg aagggttcgg cacccggaaa tttgagtcttcacctgaacc gaattctaca ggaggactta ggggtgctga caagtctgga ttgggctcctgatggtcact ttctcatctt ggccaaagca gatttgaagt tactttgcat gaagccaggggatgctccat ctgaaatctg gagcagctat acagaaaatc ctatgatatt gtccacccacaaggagtatg gcatatttgt cctgcagccc aaggatcctg gagttctttc tttcttgaggcaaaaggaat caggagagtt tgaagagagg ctgaactttg atataaactt agagaatcctagtaggaccc taatatcgat aactcaagcc aaacctgaat ctgagtcctc atttttgtgtgccagctctg atgggatcct atggaacctg gccaaatgca gcccagaagg agaatggaccacaggtaaca tgtggcagaa aaaagcaaac actccagaaa cccaaactcc agggacagacccatctacct gcagggaatc tgatgccagc atggatagtg atgccagcat ggatagtgagccaacaccac atctaaagac acggcagcgt agaaagattc actcgggctc tgtcacagccctccatgtgc tacctgagtt gctggtgaca gcttcgaagg acagagatgt taagctatgggagagaccca gtatgcagct gctgggcctg ttccgatgcg aagggtcagt gagctgcctggaaccttggc tgggcgctaa ctccaccctg cagcttgccg tgggagacgt gcagggcaatgtgtactttc tgaattggga atga Rat TEP1, Genbank #AAB51690 SEQ ID NO: 27Met Glu Lys Leu Cys Gly Tyr Val Pro Val His Pro Asp Ile Leu SerLeu Lys Asn Arg Cys Leu Thr Met Leu Ser Asp Ile Gln Pro Leu GluLys Ile His Gly Gln Arg Ser Val Asn Pro Asp Ile Leu Ser Leu GluAsn Arg Cys Leu Thr Leu Leu Pro Asp Leu Gln Pro Met Glu Lys IleHis Gly Gln Arg Ser Val His Pro Asp Ile Leu Ser Ser Glu Asn ArgCys Leu Thr Leu Leu Pro Asp Leu Gln Ser Leu Glu Lys Leu Cys GlyHis Met Ser Ser His Pro Asp Val Leu Ser Leu Glu Asn Arg Cys LeuAla Thr Leu Pro Thr Val Lys Arg Thr Val Ser Ser Gly Pro Leu LeuGln Cys Leu His Arg Ser His Thr Ala Gln Ala Asp Leu Arg Asp ProAsn Phe Arg Asn Cys Leu Phe Pro Glu Pro Pro Thr Ile Glu Ala ProCys Phe Leu Lys Glu Leu Asp Leu Pro Thr Gly Pro Arg Ala Leu LysSer Met Ser Ala Thr Ala Arg Val Gln Glu Val Ala Leu Gly Gln ArgCys Val Ser Glu Gly Lys Glu Leu Gln Glu Glu Lys Glu Ser Ala GluVal Pro Met Pro Leu Tyr Ser Leu Ser Leu Gly Gly Glu Glu Glu GluVal Val Gly Ala Pro Val Leu Lys Leu Thr Ser Gly Asp Ser Asp SerHis Pro Glu Thr Thr Asp Gln Ile Leu Gln Glu Lys Lys Met Ala LeuLeu Thr Leu Leu Cys Ser Ala Met Ala Ser Ser Val Asn Val Lys AspAla Ser Asp Pro Thr Arg Ala Ser Ile His Glu Val Cys Ser Ala LeuAla Pro Leu Glu Pro Glu Phe Ile Leu Lys Ala Ser Leu Tyr Ala ArgGln Gln Leu Asn Leu Arg Asp Ile Ala Asn Ile Val Leu Ala Val AlaAla Leu Leu Pro Ala Cys Arg Pro His Val Arg Arg Tyr Tyr Ser AlaIle Val His Leu Pro Ser Asp Trp Ile Gln Val Ala Glu Phe Tyr GlnSer Leu Ala Glu Gly Asp Glu Lys Lys Leu Val Pro Leu Pro Ala CysLeu Arg Ala Ala Met Thr Asp Lys Phe Ala Gln Phe Asp Glu Tyr GlnLeu Ala Lys Tyr Asn Pro Arg Lys His Arg Ser Lys Thr Arg Ser ArgGln Pro Pro Arg Pro Gln Arg Thr Lys Pro Pro Phe Ser Glu Ser GlyLys Cys Phe Pro Lys Ser Val Trp Pro Leu Lys Asn Glu Gln Ile SerPhe Glu Ala Ala Tyr Asn Ala Val Ser Glu Lys Lys Arg Leu Pro ArgPhe Thr Leu Lys Lys Leu Val Glu Gln Leu His Ile His Glu Pro AlaGln His Val Gln Ala Leu Leu Gly Tyr Arg Tyr Pro Ser Thr Leu GluLeu Phe Ser Arg Ser His Leu Pro Gly Pro Trp Asp Ser Ser Arg AlaGly Gln Arg Met Lys Leu Gln Arg Pro Glu Thr Trp Glu Arg Glu LeuSer Leu Arg Gly Asn Arg Ala Ser Val Trp Glu Glu Leu Ile Asp AsnGly Lys Leu Pro Phe Met Ala Met Leu Arg Asn Leu Cys Asn Leu LeuArg Thr Gly Ile Ser Ala His His His Glu Leu Val Leu Gln Arg LeuGln His Glu Lys Ser Val Ile His Ser Arg Gln Phe Pro Phe Arg PheLeu Asn Ala His Asp Ser Leu Asp Arg Leu Glu Ala Gln Leu Arg SerLys Ala Ser Pro Phe Pro Ser Asn Thr Thr Leu Met Lys Arg Ile MetIle Arg Asn Ser Lys Lys Ile Lys Arg Pro Ala Asn Pro Arg Tyr LeuCys Thr Leu Thr Gln Arg Gln Leu Arg Ala Ala Met Ala Ile Pro ValMet Tyr Glu His Leu Lys Arg Glu Lys Leu Arg Leu His Lys Ala ArgGln Trp Thr Cys Asp Leu Glu Leu Leu Glu Arg Tyr Arg Gln Ala LeuGlu Thr Ala Val Asn Ile Ser Val Lys His Asn Leu Pro Pro Leu ProGly Arg Thr Leu Leu Val Tyr Leu Thr Asp Ala Asn Ala Asn Arg LeuCys Pro Lys Ser His Leu Gln Gly Pro Pro Leu Asn Tyr Val Leu LeuLeu Ile Gly Met Met Met Ala Arg Ala Glu Gln Thr Thr Val Trp LeuCys Gly Thr Gly Thr Val Lys Thr Pro Val Leu Thr Ala Asp Glu GlyIle Leu Lys Thr Ala Ile Lys Leu Gln Ala Gln Val Gln Glu Leu GluGlu Asn Asp Glu Trp Pro Leu Glu Thr Phe Glu Lys Tyr Leu Leu SerLeu Ala Val Arg Arg Thr Pro Ile Asp Arg Val Ile Leu Phe Gly GlnArg Met Asp Thr Glu Leu Leu Asn Val Ala Lys Gln Ile Ile Trp GlnHis Val Asn Ser Lys Cys Leu Phe Val Ser Val Leu Leu Arg Lys MetGln Tyr Met Ser Pro Asn Leu Asn Pro Asn Asp Val Thr Leu Ser GlyCys Thr Asp Gly Ile Leu Lys Phe Ile Ala Glu His Gly Ala Ser ArgLeu Leu Glu His Val Gly Gln Leu Asp Lys Ile Phe Lys Ile Pro ProPro Pro Gly Lys Thr Lys Val Ser Pro Leu Arg Pro Leu Glu Glu AsnAsn Pro Gly Pro Phe Val Pro Ile Ser Gln His Gly Trp Arg Asn IleArg Leu Phe Ile Ser Ser Thr Phe Arg Asp Met His Gly Glu Arg AspLeu Leu Met Arg Ser Val Leu Pro Ala Leu Gln Ala Arg Ala Phe ProHis Arg Ile Ser Leu His Ala Ile Asp Leu Arg Trp Gly Ile Thr GluGlu Glu Thr Arg Arg Asn Arg Gln Leu Glu Val Cys Leu Gly Glu ValGlu Asn Ser Gln Leu Phe Val Gly Ile Leu Gly Ser Arg Tyr Gly TyrThr Pro Pro Ser Tyr Asp Leu Pro Asp His Pro His Phe His Trp ThrGln Arg Tyr Pro Ser Gly Arg Ser Val Thr Glu Met Glu Val MetGln Phe Leu Asn Arg Gly Gln Arg Ser Glu Pro Ser Asp Gln AlaLeu Ile Tyr Phe Arg Asp Pro Gly Phe Leu Ser Ser Val Pro AspVal Trp Lys Pro Asp Phe Ile Ser Glu Ser Glu Glu Ala Ala HisArg Val Ser Glu Leu Lys Arg Phe Leu Gln Glu Gln Lys Glu ValThr Cys Arg Arg Tyr Ser Cys Glu Trp Gly Gly Val Ala Ala GlyArg Pro Tyr Thr Gly Gly Leu Glu Glu Phe Gly Gln Leu Val LeuGln Asp Val Trp Ser Val Ile Gln Lys Arg Tyr Leu Gln Pro GlyAla Gln Leu Glu Gln Pro Gly Ser Ile Ser Glu Glu Asp Leu IleGln Ala Ser Phe Gln Gln Leu Lys Ser Pro Pro Ser Pro Ala ArgPro Arg Leu Leu Gln Asp Thr Val Gln Gln Leu Met Leu Pro HisGly Arg Leu Ser Leu Val Ile Gly Gln Ala Gly Gln Gly Lys ThrAla Phe Leu Ala Ser Leu Val Ser Ala Leu Lys Val Pro Asp GlnPro Asn Val Ala Pro Phe Val Phe Phe His Phe Ser Ala Ala ArgPro Asp Gln Cys Leu Ala Phe Asn Leu Leu Arg Arg Leu Cys ThrHis Leu His Gln Lys Leu Gly Glu Pro Ser Ala Leu Pro Ser ThrTyr Arg Gly Leu Val Trp Glu Leu Gln Gln Lys Leu Leu Leu LysSer Ala Gln Trp Leu Gln Pro Gly Gln Thr Leu Val Leu Ile IleAsp Gly Ala Asp Lys Leu Val Asp His Asn Gly Gln Leu Ile SerAsp Trp Ile Pro Lys Ser Leu Pro Arg Arg Val His Leu Val LeuSer Val Ser Ser Asp Ser Gly Leu Gly Glu Thr Leu Gln Gln SerGln Ser Ala Tyr Val Val Ala Leu Gly Ser Leu Val Pro Ser SerArg Ala Gln Leu Val Arg Glu Glu Leu Ala Leu Tyr Gly Lys ArgLeu Glu Glu Ser Pro Phe Asn Asn Gln Met Arg Leu Leu Leu AlaLys Gln Gly Ser Ser Leu Pro Leu Tyr Leu His Leu Val Thr AspTyr Leu Arg Leu Phe Thr Leu Tyr Glu Gln Val Ser Glu Arg LeuArg Thr Leu Pro Ala Thr Leu Pro Leu Leu Leu Gln His Ile LeuSer Thr Leu Glu Gln Glu His Gly His Asn Val Leu Pro Gln AlaLeu Thr Ala Leu Glu Val Thr His Ser Gly Leu Thr Val Asp GlnLeu His Ala Val Leu Ser Thr Trp Leu Thr Leu Pro Lys Glu ThrLys Ser Trp Glu Glu Ala Val Ala Ala Ser His Ser Gly Asn LeuTyr Pro Leu Ala Pro Phe Ala Tyr Leu Val Gln Ser Leu Arg SerLeu Leu Gly Glu Gly Pro Val Glu Arg Pro Gly Ala Arg Leu CysLeu Ser Asp Gly Pro Leu Arg Thr Ala Val Lys Arg Arg Tyr GlyLys Arg Leu Gly Leu Glu Lys Thr Ala His Val Leu Ile Ala AlaHis Leu Trp Lys Met Cys Asp Pro Asp Ala Ser Gly Thr Phe ArgSer Cys Pro Pro Glu Ala Leu Lys Asp Leu Pro Tyr His Leu LeuGln Ser Gly Asn His Gly Leu Leu Ala Lys Phe Leu Thr Asn LeuHis Val Val Ala Ala Tyr Leu Glu Val Gly Leu Val Pro Asp LeuLeu Glu Ala Tyr Glu Leu Tyr Ala Ser Ser Lys Pro Glu Val AsnGln Lys Leu Pro Glu Ala Asp Val Ala Val Phe His Asn Phe LeuLys Gln Gln Ala Ser Leu Leu Thr Gln Tyr Pro Leu Leu Leu LeuGln Gln Ala Ala Ser Gln Pro Glu Glu Ser Pro Val Cys Cys GlnAla Pro Leu Leu Thr Gln Arg Trp His Asn Gln Cys Ile Leu LysTrp Ile Asn Lys Pro Gln Thr Leu Lys Gly Gln Gln Ser Leu SerLeu Pro Ile Ser Ser Ser Pro Thr Ala Val Ala Phe Ser Pro AsnGly Gln Arg Ala Ala Val Gly Thr Ala Gly Gly Thr Ile Tyr LeuLeu Asn Leu Arg Thr Trp Gln Glu Glu Lys Ala Leu Val Ser GlyCys Asp Gly Ile Ser Ser Phe Ala Phe Leu Ser Asp Thr Ala LeuPhe Leu Thr Thr Phe Asp Gly Leu Leu Glu Leu Trp Asp Leu GlnHis Gly Cys Trp Val Phe Gln Thr Lys Ala His Gln Tyr Gln IleThr Gly Cys Cys Leu Ser Pro Asp Arg Arg Leu Leu Ala Thr ValCys Leu Gly Gly Tyr Val Lys Leu Trp Asp Thr Val Gln Gly GlnLeu Ala Phe Gln Tyr Thr His Pro Lys Ser Leu Asn Cys Ile ThrPhe His Pro Glu Gly Gln Val Val Ala Thr Gly Asn Trp Ser GlyIle Val Thr Phe Phe Gln Ala Asp Gly Leu Lys Val Thr Lys GluLeu Gly Gly Pro Gly Pro Ser Val Arg Thr Leu Ala Phe Ser AlaPro Gly Lys Val Val Ala Leu Gly Arg Ile Asp Gly Thr Val GluLeu Trp Ala Trp Gln Glu Gly Thr Arg Leu Ala Ala Phe Pro AlaGln Cys Gly Gly Val Ser Thr Val Leu Phe Leu His Ala Gly GlyArg Phe Leu Thr Ala Gly Glu Asp Gly Lys Ala Gln Leu Trp SerGly Phe Leu Gly Arg Pro Arg Gly Cys Leu Gly Ser Leu Tyr LeuSer Pro Ala Leu Ser Val Ala Leu Asn Pro Asp Gly Asp Gln ValAla Val Gly Tyr Arg Gly Asp Gly Ile Lys Ile Tyr Arg Ile SerSer Gly Pro Gln Glu Ala Gln Cys Gln Glu Leu Asn Val Ala ValSer Ala Leu Val Trp Leu Ser Pro Ser Val Leu Val Ser Gly AlaGlu Asp Gly Ser Leu His Gly Trp Met Leu Arg Arg Asn Ser LeuGln Ser Leu Trp Leu Ser Ser Val Cys Gln Lys Pro Val Leu GlyLeu Ala Ala Ser Gln Glu Phe Leu Ala Ser Ala Ser Glu Asp PheThr Val Arg Leu Trp Pro Arg Gln Leu Leu Thr Gln Pro His AlaVal Glu Glu Leu Pro Cys Ala Ala Glu Leu Arg Gly His Glu GlyPro Val Cys Cys Cys Ser Phe Ser Pro Asp Gly Arg Ile Leu AlaThr Ala Gly Arg Asp Arg Asn Leu Leu Cys Trp Asp Val Lys ValAla Gln Ala Pro Leu Leu Ile His Thr Phe Ser Ser Cys His ArgAsp Trp Ile Thr Gly Cys Thr Trp Thr Lys Asp Asn Ile Leu IleSer Cys Ser Ser Asp Gly Ser Val Gly Leu Trp Asn Pro Glu AlaGly Gln Gln Leu Gly Gln Phe Pro Gly His Gln Ser Ala Val SerAla Val Val Ala Val Glu Glu His Ile Val Ser Val Ser Arg AspGly Thr Leu Lys Val Trp Asp Arg Gln Gly Val Glu Leu Thr SerIle Pro Ala His Ser Gly Pro Ile Ser Gln Cys Ala Ala Ala LeuGlu Pro Arg Pro Ala Gly Gln Pro Gly Ser Glu Leu Met Val ValThr Val Gly Leu Asp Gly Ala Thr Lys Leu Trp His Pro Leu LeuVal Cys Gln Ile His Thr Leu Gln Gly His Ser Gly Pro Val ThrAla Ala Ala Ala Ser Glu Ala Ser Gly Leu Leu Leu Thr Ser AspAsn Ser Ser Val Arg Leu Trp Gln Ile Pro Lys Glu Ala Asp AspThr Cys Lys Pro Arg Ser Ser Ala Val Ile Thr Ala Val Ala TrpAla Pro Asp Gly Ser Leu Val Val Ser Gly Asn Glu Ala Gly GluLeu Thr Leu Trp Gln Lys Ala Gln Ala Val Ala Thr Ala Arg AlaPro Gly Arg Val Ser Asp Leu Ile Trp Cys Ser Ala Asn Ala PhePhe Val Leu Ser Ala Asn Glu Asn Val Ser Glu Trp Gln Val GluLeu Arg Lys Gly Ser Thr Cys Thr Asn Phe Arg Leu Tyr Leu LysArg Val Leu Gln Glu Asp Leu Gly Val Leu Thr Gly Met Ala LeuAla Pro Asp Gly Gln Ser Leu Ile Leu Met Lys Glu Asp Val GluLeu Leu Gln Met Lys Pro Gly Ser Thr Pro Ser Ser Ile Cys ArgArg Tyr Ala Val His Ser Ser Ile Leu Cys Thr Ser Lys Asp TyrGly Leu Phe Tyr Leu Gln Gln Gly Asn Ser Gly Ser Leu Ser IleLeu Glu Gln Glu Glu Ser Gly Lys Phe Glu Lys Thr Leu Asp PheAsn Leu Asn Leu Asn Asn Pro Asn Gly Ser Pro Val Ser Ile ThrGln Ala Glu Pro Glu Ser Gly Ser Ser Leu Leu Cys Ala Thr SerAsp Gly Met Leu Trp Asn Leu Ser Glu Cys Thr Pro Glu Gly GluTrp Val Val Asp Asn Ile Trp Gln Lys Lys Ser Arg Asn Pro LysSer Arg Thr Pro Gly Thr Asp Ser Ser Pro Gly Leu Phe Cys MetAsp Ser Trp Val Glu Pro Thr His Leu Lys Ala Arg Gln Cys LysLys Ile His Leu Gly Ser Val Thr Ala Leu His Val Leu Pro GlyLeu Leu Val Thr Ala Ser Glu Asp Arg Asp Val Lys Leu Trp GluArg Pro Ser Met Gln Leu Leu Gly Leu Phe Arg Cys Glu Gly ProVal Ser Cys Leu Glu Pro Trp Met Glu Pro Ser Ser Pro Leu GlnLeu Ala Val Gly Asp Ala Gln Gly Asn Leu Tyr Phe Leu Ser Trp GluRat TEP1 cDNA, Genbank #U89282 SEQ ID NO: 28atggagaaac tctgtggtta tgtgcctgtc cacccagaca tcctctcctt gaagaatcggtgcctgacca tgctctctga catccaaccc ctggagaaaa tacatggaca gagatctgtcaacccagaca tcctgtcctt ggagaaccgg tgcctgacct tgctccctga tctccagcccatggagaaaa tacatggaca gagatctgtc cacccagaca tcctctcctc agagaaccggtgtctgacct tgctccctga cctccagtcc ctggagaagc tatgtggaca tatgtctagtcacccagacg tcctctcttt ggagaaccga tgtcttgcta ccctcccgac tgtaaagagaactgtttcga gtggcccctt gctccagtgt cttcacagat ctcatacggc acaagctgatctgcgtgacc cgaactttcg caactgcctg ttccctgagc ctcctaccat agaggctccatgtttcttga aggaactaga ccttccaact ggacccaggg ccctgaaatc catgtctgctacagctcgag ttcaggaagt agctttgggt cagcggtgcg tctcagaagg aaaggaattgcaggaagaaa aagaaagcgc agaagtcccg atgcctttgt acagtctaag cttggggggagaagaagaag aagtggtggg ggcaccggtc ctaaaactca catctggaga ctctgactctcaccctgaaa ccactgacca gatcctgcag gagaagaaga tggctctctt gaccttgctgtgctcagcta tggcctcaag tgtgaatgtg aaagatgcct ccgatcctac ccgggcatctatccatgaag tctgcagtgc gctggccccc ttggaacctg agttcatcct taaggcatctttgtatgcta ggcagcagct taacctccgg gacatagcca atatagtgtt ggccgtggctgccctcttgc cagcctgccg cccccatgta cgacggtatt actctgccat tgttcacctgccttcagact ggatccaggt agccgagttc taccagagcc tggcagaagg ggatgagaagaagttggtgc ccctgcctgc ctgcctccgt gctgccatga ctgacaaatt tgcccagtttgatgagtacc agctagcgaa gtacaaccca cggaaacacc gatccaagac acgttcccgccagccacccc gccctcaaag gacaaaacct ccattttcag agagtgggaa atgttttccaaagagcgttt ggccccttaa aaacgaacag atttcgttcg aagcagctta taatgcagtgtcagagaaga aaaggctacc aaggttcact ctgaagaagt tggtagagca actgcatatccatgagcctg cgcagcatgt ccaggccctg ctgggctaca ggtacccatc caccctagagctcttttctc ggagtcatct ccctgggcca tgggactcta gcagggctgg gcaacggatgaagctccaaa ggccagagac ctgggagcgg gagctgagct tacgtggaaa cagagcttctgtgtgggagg aactcataga caatgggaaa ctccccttca tggccatgct ccggaacctgtgtaacctgc tgcggactgg gatcagtgcc caccaccatg aactcgttct ccagagactccagcatgaga aatctgtgat tcacagtcgg cagtttccat tcagattcct taatgctcacgactctctcg atagactcga ggctcagctc agaagtaaag catcgccctt cccttccaatacaacattga tgaagcggat aatgattaga aactcaaaaa aaatcaagag acctgccaacccgaggtacc tgtgcaccct gacgcagcgg cagcttcggg cggcaatggc tatcccggtgatgtatgagc atctcaagcg ggagaaactg aggctgcaca aggccagaca gtggacctgtgaccttgagt tgctggagcg gtatcgccag gccctggaaa cggccgtgaa catctctgtaaagcacaacc tacccccgct gccaggccga accctcttgg tctatctcac agatgcaaatgccaacagac tttgtcccaa gagtcacttg caagggcctc ccctgaacta tgtgctgctgttgatcggga tgatgatggc tcgggcggag cagacgacag tttggctgtg tgggacaggaactgtgaaga caccagtact tacagccgac gaaggtatcc tgaagactgc catcaaacttcaggctcaag tccaggagtt agaagaaaat gatgagtggc ccctggaaac ttttgagaagtacctgctat ctctggctgt gcgaaggacc cctattgaca gggtcatcct gttcggccaaaggatggata cggagctgct gaatgtagcc aaacagatta tctggcagca tgtgaattccaagtgcctct tcgtcagtgt cctcctacgg aaaatgcagt acatgtcacc aaatttgaatcccaatgatg tgacgctctc gggctgcact gacgggatcc tgaagttcat tgcggagcatggagcctctc gtcttctgga acatgtgggc caactagata agatattcaa gatccctccacccccaggaa agacaaaggt ctcacctctc cggccgctgg aggagaacaa ccctggtcccttcgttccta tttcccagca tggatggcgc aacatccggc ttttcatttc gtccactttccgagacatgc atggggaacg agacttgctg atgcgatctg ttctgccagc gctgcaggcccgagcgttcc cccaccgcat cagccttcac gccattgacc tgcgctgggg aatcacggaggaagagaccc gcaggaacag acaactggaa gtgtgccttg gggaggtgga gaactctcagctgttcgtgg ggatcctggg ctcccgctat ggctatactc cccccagcta tgatctgcctgaccaccccc actttcactg gacccagcga tacccttcgg ggcgctctgt aacagagatggaggtgatgc agttcctgaa ccgtggccaa cgctcggaac cctctgacca agctctcatctacttccgag atcctggttt ccttagctct gtgccagatg tctggaaacc tgactttatttccgagtcag aagaggctgc acatcgggtc tcagaactga agagattcct acaggaacagaaagaggtta cctgccgcag gtactcctgt gaatggggag gcgtagcagc cggccggccctatactgggg gcctggagga gtttggacag ttggttctcc aagatgtgtg gagcgtgatccagaagcgtt acctgcagcc tggggcccag ttggagcagc caggatccat ctcagaagaggatttgatcc aggccagctt tcagcagctg aagagcccac cgagtcccgc acggccacgccttcttcagg ataccgtgca acagctgatg ctgccccacg ggaggctgag cctagtgattgggcaggcag gacagggaaa gactgccttc ctggcatccc ttgtgtcggc cctgaaggttcccgaccagc ccaatgtggc cccgttcgtt ttcttccact tttcagcagc ccgccctgaccagtgtcttg ctttcaacct cctcagacgc ctctgtaccc atctgcatca aaaactgggagagccgagcg ctctccccag cacttacaga ggcctggtgt gggaactgca gcagaagctgctcctcaaat ctgcccagtg gctgcaacca ggccagactt tggtccttat tatcgacggggcagataagt tggtggacca taatggacag ctgatttcag actggatccc caagtctcttccgcggcgag tacacctggt gctgagtgtg tctagtgact caggcctggg agagacccttcagcaaagtc agagtgctta tgtggtggcc ttggggtctt tggtcccgtc ttcaagggctcagcttgtga gagaagagct agcactgtat gggaaacggc tggaggagtc accttttaacaaccagatgc ggctgctgct ggcaaagcag gggtcaagcc tgccactgta cctgcacctcgtcactgact acctgaggct tttcacactg tacgaacagg tgtctgagag acttcgaaccctgcccgcca ctctcccact gctgctgcag cacatcctga gcaccttgga gcaagagcatggccataacg tccttcctca agctttgact gcccttgagg tcacgcacag tggtctgactgtggaccagc tgcatgcagt cctgagcacg tggttgactt tgcccaagga gactaagagctgggaagagg cagtggctgc cagtcacagt ggaaacctct accccttggc tccatttgcctaccttgtcc agagtctacg cagtttacta ggcgagggcc ccgtggagcg ccctggcgcccgtctctgcc tctctgatgg gcctctgagg acagcagtta aacgtcgcta tgggaaaaggctggggctag agaagactgc gcatgtcctc attgcagctc acctctggaa gatgtgtgaccctgatgcct caggcacctt ccgaagttgc cctcccgagg ctctgaaaga tttaccttaccacctgctcc agagcgggaa ccatggtctc cttgcaaagt tccttaccaa cctccatgtggtggctgcat atctggaagt gggtctagtc ccggacctct tggaggctta cgagctctatgcttcttcaa agcctgaagt gaaccagaag ctcccggagg cagatgttgc tgtattccacaacttcctga aacaacaggc ttcactcctt acccagtatc ctttgctcct gctccagcaggcagctagcc agcctgaaga gtcacctgtt tgctgccagg cccccctgct cacccagcggtggcacaacc agtgcatact gaaatggatt aataaacccc agaccttgaa gggtcagcaaagcttgtctc tgccaatttc ctcatcccca actgctgtgg ccttctctcc taatgggcaaagagcagctg tggggactgc tggtgggaca atttacctgt tgaacttgag aacctggcaggaggagaagg ctctggtgag tggctgtgat gggatttcct ctttcgcgtt cctgtcagacactgctcttt tccttaccac cttcgatggg ctcctggagc tttgggacct gcaacatggttgttgggtgt tccagaccaa ggcccaccag taccaaatca ctggctgctg cctgagcccagaccgccgcc tgctggccac cgtgtgtttg ggaggatacg taaagctgtg ggacacagtccagggccagc tggctttcca gtacacccat cccaagtctc taaactgcat caccttccacccagaggggc aggtggtagc cacaggcaac tggtctggca tcgtgacctt cttccaggcagatggactca aagtcaccaa ggaactaggg ggcccaggac cctctgttcg tacgctggcattcagtgcac ccgggaaggt tgtggctcta ggccggatag atgggacagt ggagctgtgggcctggcaag agggcacacg gctggcagcc ttccctgcac agtgtggcgg tgtctccaccgttcttttct tgcatgctgg aggccggttc ctgacggctg gggaagatgg caaggctcagttatggtcag gatttcttgg ccggcccagg ggttgcctgg gctctcttta tctttctcctgcgctctctg tggctctcaa cccagacggt gaccaggtgg ctgttgggta ccgaggagatggcattaaaa tctacagaat ttcttcaggt ccccaggagg ctcaatgcaa agagctaaatgtggcggtgt ctgcactggt ctggctgagt cccagcgtct tggtgagtgg tgcagaagatggctccctgc atggctggat gctcaggaga aactcccttc agtccctgtg gctgtcatccgtgtgccaga agcctgtgct ggggctggct gcctcccagg agttcttggc ttctgcctcagaggacttca cggtgcgact gtggccaaga cagctgctga cacagccaca tgcagtagaagagttgccct gtgcggctga actccgggga cacgaggggc cggtgtgctg ctgtagcttcagcccggatg gacgcatctt ggccacagcg ggcagggatc ggaatctcct ctgctgggacgtcaaggtag cccaagcccc tctcctgatt cacacgttct cgtcctgtca tcgagactggatcactggct gtacgtggac caaagacaac atcctgatct cctgctctag tgatggctctgtgggactct ggaacccaga ggcaggacag caacttggcc agttcccagg tcaccagagtgccgtgagcg ctgtggttgc tgtggaggaa cacattgtat ctgtgagtcg ggatgggaccttgaaagtgt gggaccgtca gggtgtggag ctgaccagca tccctgccca ttccggacccattagccagt gtgcggctgc tctggaaccc cgtccagctg gacagcctgg atcagagcttatggtggtga ctgttggact ggatggggcc acaaagctgt ggcatcccct gttggtgtgccaaatacata ccctgcaggg acacagtggt ccagtcacag ctgctgctgc ttcagaggcctcaggcctcc tgctgacctc agacaatagc tctgtacgac tctggcagat ccctaaggaagcagatgata cctgcaaacc taggagttct gcggtcatca ccgctgtggc gtgggcaccagatggttctc tggtggtgtc tggaaatgaa gctggggaac taacgctgtg gcagaaagcgcaggctgtgg ctacggcacg ggctccaggc cgcgtcagtg acctgatctg gtgctccgcaaatgcattct ttgttctcag tgctaatgaa aatgtcagtg agtggcaagt ggaactgaggaaaggttcaa catgcaccaa tttcagactt tatctgaaga gagttctgca ggaggacttgggagtcttga caggtatggc cctggcgcct gacggccagt ctctcatttt gatgaaagaggatgtagaat tgctacagat gaagcccggg tctactccat cttcgatctg caggaggtatgcagtgcatt cttctatact gtgcaccagc aaagactatg gcctgtttta cctgcagcagggaaactctg gatctctttc tatcttggag caggaggagt cagggaagtt tgaaaagaccctggacttca atctgaactt aaataatcct aatgggtccc cagtatcaat cactcaggctgaacctgagt ctgggtcctc gcttttgtgt gctacctctg atgggatgct gtggaacttatctgagtgta ccccagaagg agagtgggtc gtagataaca tctggcagaa aaaatcaagaaaccctaaaa gtcgaactcc ggggacagat tcgtccccag gcttattctg catggatagctgggtagaac ccacacattt aaaggcacgg cagtgtaaaa agattcactt gggctctgtcacggccctcc atgtgctgcc cggattgctg gtgactgctt cagaggacag agatgttaagctgtgggaga gacccagtat gcagctgctc ggcttgttcc gatgtgaagg gccggtgagctgtctggaac cttggatgga gcccagctct cccctgcagc ttgctgtggg agatgcacaaggaaacttgt attttctatc ttgggaatga Human vRNA, Genbank #AF045143SEQ ID NO: 29ggcuggcuuu agcucagcgg uuacuucgac aguucuuuaa uugaaacaag caaccugucuggguuguucg agacccgcgg gcgcucucca gcuuuuuu Human vRNA, Genbank #AF045144SEQ ID NO: 30ggcuggcuuu agcucagcgg uuacuucgag uacauuguaa ccaccucucu gggugguucgagacccgcgg gugcuuucca gcucuuuu Human vRNA, Genbank #AF045145SEQ ID NO: 31ggcuggcuuu agcucagcgg uuacuucgcg ugucaucaaa ccaccucucu ggguuguucgagacccgcgg gcgcucucca gcccucuu Rat vRNA, Genbank #Z1171 SEQ ID NO: 32ggccagcuuu agcucagcgg uuacuucgac gugcuccagu uugagcaggc uauguaacguggucgguucg agcaacacaa ccagccgcuu gccuaucugg ugagugguug guucgagacccgcgggcgcu cucuggcccu uuuHuman IL-2 cDNA Sequence, Genbank #BC070338.1, coding sequence: 48-509SEQ ID NO: 33 1atcactctct ttaatcacta ctcacagtaa cctcaactcc tgccacaatg tacaggatgc 61aactcctgtc ttgcattgca ctaagtcttg cacttgtcac aaacagtgca cctacttcaa 121gttctacaaa gaaaacacag ctacaactgg agcatttact gctggattta cagatgattt 181tgaatggaat taataattac aagaatccca aactcaccag gatgctcaca tttaagtttt 241acatgcccaa gaaggccaca gaactgaaac atcttcagtg tctagaagaa gaactcaaac 301ctctggagga agtgctaaat ttagctcaaa gcaaaaactt tcacttaaga cccagggact 361taatcagcaa tatcaacgta atagttctgg aactaaaggg atctgaaaca acattcatgt 421gtgaatatgc tgatgagaca gcaaccattg tagaatttct gaacagatgg attacctttt 481gtcaaagcat catctcaaca ctgacttgat aattaagtgc ttcccactta aaacgtatca 541ggccttctat ttatttaaat atttaaattt tatatttatt gttgaatgta tggtttgcta 601cctattgtaa ctattattct taatcttaaa actataaata tggatctttt atgattcttt 661ttgtaagccc taggggctct aaaatggttt cacttattta tcccaaaata tttattatta 721tgttgaatgt taaatatagt atctatgtag attggttagt aaaactattt aataaatttg 781ataaatataa aaaaaaaaaa aaaaaaaaaa aaaaHuman IL-2 Protein sequence, Genbank #AAH70338.1 SEQ ID NO: 34MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRFRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTHuman IL-7 cDNA sequence, Genbank #J04156.1; coding sequence: 385-918SEQ ID NO: 35 1gaattcctct ggtcctcatc caggtgcgcg ggaagcaggt gcccaggaga gaggggataa 61tgaagattcc atgctgatga tcccaaagat tgaacctgca gaccaagcgc aaagtagaaa 121ctgaaagtac actgctggcg gatcctacgg aagttatgga aaaggcaaag cgcagagcca 181cgccgtagtg tgtgccgccc cccttgggat ggatgaaact gcagtcgcgg cgtgggtaag 241aggaaccagc tgcagagatc accctgccca acacagactc ggcaactccg cggaagacca 301gggtcctggg agtgactatg ggcggtgaga gcttgctcct gctccagttg cggtcatcat 361gactacgccc gcctcccgca gaccatgttc catgtttctt ttaggtatat ctttggactt 421cctcccctga tccttgttct gttgccagta gcatcatctg attgtgatat tgaaggtaaa 481gatggcaaac aatatgagag tgttctaatg gtcagcatcg atcaattatt ggacagcatg 541aaagaaattg gtagcaattg cctgaataat gaatttaact tttttaaaag acatatctgt 601gatgctaata aggaaggtat gtttttattc cgtgctgctc gcaagttgag gcaatttctt 661aaaatgaata gcactggtga ttttgatctc cacttattaa aagtttcaga aggcacaaca 721atactgttga actgcactgg ccaggttaaa ggaagaaaac cagctgccct gggtgaagcc 781caaccaacaa agagtttgga agaaaataaa tctttaaagg aacagaaaaa actgaatgac 841ttgtgtttcc taaagagact attacaagag ataaaaactt gttggaataa aattttgatg 901ggcactaaag aacactgaaa aatatggagt ggcaatatag aaacacgaac tttagctgca 961tcctccaaga atctatctgc ttatgcagtt tttcagagtg gaatgcttcc tagaagttac 1021tgaatgcacc atggtcaaaa cggattaggg catttgagaa atgcatattg tattactaga 1081agatgaatac aaacaatgga aactgaatgc tccagtcaac aaactatttc ttatatatgt 1141gaacatttat caatcagtat aattctgtac tgatttttgt aagacaatcc atgtaaggta 1201tcagttgcaa taatacttct caaacctgtt taaatatttc aagacattaa atctatgaag 1261tatataatgg tttcaaagat tcaaaattga cattgcttta ctgtcaaaat aattttatgg 1321ctcactatga atctattata ctgtattaag agtgaaaatt gtcttcttct gtgctggaga 1381tgttttagag ttaacaatga tatatggata atgccggtga gaataagaga gtcataaacc 1441ttaagtaagc aacagcataa caaggtccaa gatacctaaa agagatttca agagatttaa 1501ttaatcatga atgtgtaaca cagtgccttc aataaatggt atagcaaatg ttttgacatg 1561aaaaaaggac aatttcaaaa aaataaaatHuman IL-7 Protein sequence, Genbank #AAA59156.1 SEQ ID NO: 36MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKFAALGEAQFTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHHuman IL-15 cDNA sequence, Genbank #BC018149.2; coding sequence: 845-1333SEQ ID NO: 37 1actccgggtg gcaggcgccc gggggaatcc cagctgactc gctcactgcc ttcgaagtcc 61ggcgcccccc gggagggaac tgggtggccg caccctcccg gctgcggtgg ctgtcgcccc 121ccaccctgca gccaggactc gatggaggta cagagctcgg cttctttgcc ttgggagggg 181agtggtggtg gttgaaaggg cgatggaatt ttccccgaaa gcctacgccc agggcccctc 241ccagctccag cgttaccctc cggtctatcc tactggccga gctgccccgc cttctcatgg 301ggaaaactta gccgcaactt caatttttgg tttttccttt aatgacactt ctgaggctct 361cctagccatc ctcccgcttc cggaggagcg cagatcgcag gtccctttgc ccctggcgtg 421cgactcccta ctgcgctgcg ctcttacggc gttccaggct gctggctagc gcaaggcggg 481ccgggcaccc cgcgctccgc tgggagggtg agggacgcgc gtctggcggc cccagccaag 541ctgcgggttt ctgagaagac gctgtcccgc agccctgagg gctgagttct gcacccagtc 601aagctcagga aggccaagaa aagaatccat tccaatatat ggccatgtgg ctctttggag 661caatgttcca tcatgttcca tgctgctgac gtcacatgga gcacagaaat caatgttagc 721agatagccag cccatacaag atcgtattgt attgtaggag gcatcgtgga tggatggctg 781ctggaaaccc cttgccatag ccagctcttc ttcaatactt aaggatttac cgtggctttg 841agtaatgaga atttcgaaac cacatttgag aagtatttcc atccagtgct acttgtgttt 901acttctaaac agtcattttc taactgaagc tggcattcat gtcttcattt tgggctgttt 961cagtgcaggg cttcctaaaa cagaagccaa ctgggtgaat gtaataagtg atttgaaaaa 1021aattgaagat cttattcaat ctatgcatat tgatgctact ttatatacgg aaagtgatgt 1081tcaccccagt tgcaaagtaa cagcaatgaa gtgctttctc ttggagttac aagttatttc 1141acttgagtcc ggagatgcaa gtattcatga tacagtagaa aatctgatca tcctagcaaa 1201caacagtttg tcttctaatg ggaatgtaac agaatctgga tgcaaagaat gtgaggaact 1261ggaggaaaaa aatattaaag aatttttgca gagttttgta catattgtcc aaatgttcat 1321caacacttct tgattgcaat tgattctttt taaagtgttt ctgttattaa caaacatcac 1381tctgctgctt agacataaca aaacactcgg catttcaaat gtgctgtcaa aacaagtttt 1441tctgtcaaga agatgatcag accttggatc agatgaactc ttagaaatga aggcagaaaa 1501atgtcattga gtaatatagt gactatgaac ttctctcaga cttactttac tcattttttt 1561aatttattat tgaaattgta catatttgtg gaataatgta aaatgttgaa taaaaatatg 1621tacaagtgtt gttttttaag ttgcactgat attttacctc ttattgcaaa atagcatttg 1681tttaagggtg atagtcaaat tatgtattgg tggggctggg taccaatgct gcaggtcaac 1741agctatgctg gtaggctcct gcctgtgtgg aaccactgac tactggctct cattgacttc 1801cttactaagc atagcaaaca gaggaagaat ttgttatcag taagaaaaag aagaactata 1861tgtgaatcct cttctttaca ctgtaattta gttattgatg tataaagcaa ctgttatgaa 1921ataaagaaat tgcaataact ggcaaaaaaa aaaaaaaaaa aaaaaaaaHuman IL-15 protein sequence, Genbank #AAH18149.1 SEQ ID NO: 38MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINHuman IL-12B cDNA Sequence, Genbank #NM_002187.2; coding sequence: 43-1029SEQ ID NO: 39 1ctgtttcagg gccattggac tctccgtcct gcccagagca agatgtgtca ccagcagttg 61gtcatctctt ggttttccct ggtttttctg gcatctcccc tcgtggccat atgggaactg 121aagaaagatg tttatgtcgt agaattggat tggtatccgg atgcccctgg agaaatggtg 181gtcctcacct gtgacacccc tgaagaagat ggtatcacct ggaccttgga ccagagcagt 241gaggtcttag gctctggcaa aaccctgacc atccaagtca aagagtttgg agatgctggc 301cagtacacct gtcacaaagg aggcgaggtt ctaagccatt cgctcctgct gcttcacaaa 361aaggaagatg gaatttggtc cactgatatt ttaaaggacc agaaagaacc caaaaataag 421acctttctaa gatgcgaggc caagaattat tctggacgtt tcacctgctg gtggctgacg 481acaatcagta ctgatttgac attcagtgtc aaaagcagca gaggctcttc tgacccccaa 541ggggtgacgt gcggagctgc tacactctct gcagagagag tcagagggga caacaaggag 601tatgagtact cagtggagtg ccaggaggac agtgcctgcc cagctgctga ggagagtctg 661cccattgagg tcatggtgga tgccgttcac aagctcaagt atgaaaacta caccagcagc 721ttcttcatca gggacatcat caaacctgac ccacccaaga acttgcagct gaagccatta 781aagaattctc ggcaggtgga ggtcagctgg gagtaccctg acacctggag tactccacat 841tcctacttct ccctgacatt ctgcgttcag gtccagggca agagcaagag agaaaagaaa 901gatagagtct tcacggacaa gacctcagcc acggtcatct gccgcaaaaa tgccagcatt 961agcgtgcggg cccaggaccg ctactatagc tcatcttgga gcgaatgggc atctgtgccc 1021tgcagttagg ttctgatcca ggatgaaaat ttggaggaaa agtggaagat attaagcaaa 1081atgtttaaag acacaacgga atagacccaa aaagataatt tctatctgat ttgctttaaa 1141acgttttttt aggatcacaa tgatatcttt gctgtatttg tatagttaga tgctaaatgc 1201tcattgaaac aatcagctaa tttatgtata gattttccag ctctcaagtt gccatgggcc 1261ttcatgctat ttaaatattt aagtaattta tgtatttatt agtatattac tgttatttaa 1321cgtttgtctg ccaggatgta tggaatgttt catactctta tgacctgatc catcaggatc 1381agtccctatt atgcaaaatg tgaatttaat tttatttgta ctgacaactt ttcaagcaag 1441gctgcaagta catcagtttt atgacaatca ggaagaatgc agtgttctga taccagtgcc 1501atcatacact tgtgatggat gggaacgcaa gagatactta catggaaacc tgacaatgca 1561aacctgttga gaagatccag gagaacaaga tgctagttcc catgtctgtg aagacttcct 1621ggagatggtg ttgataaagc aatttagggc cacttacact tctaagcaag tttaatcttt 1681ggatgcctga attttaaaag ggctagaaaa aaatgattga ccagcctggg aaacataaca 1741agaccccgtc tctacaaaaa aaatttaaaa ttagccaggc gtggtggctc atgcttgtgg 1801tcccagctgt tcaggaggat gaggcaggag gatctcttga gcccaggagg tcaaggctat 1861ggtgagccgt gattgtgcca ctgcatacca gcctaggtga cagaatgaga ccctgtctca 1921aaaaaaaaaa tgattgaaat taaaattcag ctttagcttc catggcagtc ctcaccccca 1981cctctctaaa agacacagga ggatgacaca gaaacaccgt aagtgtctgg aaggcaaaaa 2041gatcttaaga ttcaagagag aggacaagta gttatggcta aggacatgaa attgtcagaa 2101tggcaggtgg cttcttaaca gccctgtgag aagcagacag atgcaaagaa aatctggaat 2161ccctttctca ttagcatgaa tgaacctgat acacaattat gaccagaaaa tatggctcca 2221tgaaggtgct acttttaagt aatgtatgtg cgctctgtaa agtgattaca tttgtttcct 2281gtttgtttat ttatttattt atttttgcat tctgaggctg aactaataaa aactcttctt 2341tgtaatc Human IL-12B Protein Sequence, Genbank #NP_002178.2SEQ ID NO: 40MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSHuman IL-12A cDNA sequence, Genbank #NM_000882.2; coding sequence: 216-977SEQ ID NO: 41 1tttcattttg ggccgagctg gaggcggcgg ggccgtcccg gaacggctgc ggccgggcac 61cccgggagtt aatccgaaag cgccgcaagc cccgcgggcc ggccgcaccg cacgtgtcac 121cgagaagctg atgtagagag agacacagaa ggagacagaa agcaagagac cagagtcccg 181ggaaagtcct gccgcgcctc gggacaatta taaaaatgtg gccccctggg tcagcctccc 241agccaccgcc ctcacctgcc gcggccacag gtctgcatcc agcggctcgc cctgtgtccc 301tgcagtgccg gctcagcatg tgtccagcgc gcagcctcct ccttgtggct accctggtcc 361tcctggacca cctcagtttg gccagaaacc tccccgtggc cactccagac ccaggaatgt 421tcccatgcct tcaccactcc caaaacctgc tgagggccgt cagcaacatg ctccagaagg 481ccagacaaac tctagaattt tacccttgca cttctgaaga gattgatcat gaagatatca 541caaaagataa aaccagcaca gtggaggcct gtttaccatt ggaattaacc aagaatgaga 601gttgcctaaa ttccagagag acctctttca taactaatgg gagttgcctg gcctccagaa 661agacctcttt tatgatggcc ctgtgcctta gtagtattta tgaagacttg aagatgtacc 721aggtggagtt caagaccatg aatgcaaagc ttctgatgga tcctaagagg cagatctttc 781tagatcaaaa catgctggca gttattgatg agctgatgca ggccctgaat ttcaacagtg 841agactgtgcc acaaaaatcc tcccttgaag aaccggattt ttataaaact aaaatcaagc 901tctgcatact tcttcatgct ttcagaattc gggcagtgac tattgataga gtgatgagct 961atctgaatgc ttcctaaaaa gcgaggtccc tccaaaccgt tgtcattttt ataaaacttt 1021gaaatgagga aactttgata ggatgtggat taagaactag ggagggggaa agaaggatgg 1081gactattaca tccacatgat acctctgatc aagtattttt gacatttact gtggataaat 1141tgtttttaag ttttcatgaa tgaattgcta agaagggaaa atatccatcc tgaaggtgtt 1201tttcattcac tttaatagaa gggcaaatat ttataagcta tttctgtacc aaagtgtttg 1261tggaaacaaa catgtaagca taacttattt taaaatattt atttatataa cttggtaatc 1321atgaaagcat ctgagctaac ttatatttat ttatgttata tttattaaat tatttatcaa 1381gtgtatttga aaaatatttt taagtgttct aaaaataaaa gtattgaatt aaagtgaaaa 1441aaaa Human IL-12A Protein sequence, Genbank #NP_000873.2 SEQ ID NO: 42MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASHuman CSF2 cDNA Sequence, Genbank #BC108724.1; coding sequence: 20-454SEQ ID NO: 43 1ggctaaagtt ctctggagga tgtggctgca gagcctgctg ctcttgggca ctgtggcctg 61cagcatctct gcacccgccc gctcgcccag ccccagcacg cagccctggg agcatgtgaa 121tgccatccag gaggcccggc gtctcctgaa cctgagtaga gacactgctg ctgagatgaa 181tgaaacagta gaagtcatct cagaaatgtt tgacctccag gagccgacct gcctacagac 241ccgcctggag ctgtacaagc agggcctgcg gggcagcctc accaagctca agggcccctt 301gaccatgatg gccagccact acaagcagca ctgccctcca accccggaaa cttcctgtgc 361aacccagatt atcacctttg aaagtttcaa agagaacctg aaggactttc tgcttgtcat 421cccctttgac tgctgggagc cagtccagga gtgagaccgg ccagatgagg ctggccaagc 481cggggagctg ctctctcatg aaacaagagc tagaaactca ggatggtcat cttggaggga 541ccaaggggtg ggccacagcc atggtgggag tggcctggac ctgccctggg ccacactgac 601cctgatacag gcatggcaga agaatgggaa tattttatac tgacagaaat cagtaatatt 661tatatattta tatttttaaa atatttattt atttatttat ttaagttcat attccatatt 721tattcaagat gttttaccgt aataattatt attaaaaata tgcttctact tgaaaaaaaa 781aaaaaaa Human CSF2 Protein Sequence, Genbank #AAI08725.1 SEQ ID NO: 44MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQEHuman CXCL9 cDNA Sequence, Genbank #NM_002416.1; coding sequence: 40-417SEQ ID NO: 45 1atccaataca ggagtgactt ggaactccat tctatcacta tgaagaaaag tggtgttctt 61ttcctcttgg gcatcatctt gctggttctg attggagtgc aaggaacccc agtagtgaga 121aagggtcgct gttcctgcat cagcaccaac caagggacta tccacctaca atccttgaaa 181gaccttaaac aatttgcccc aagcccttcc tgcgagaaaa ttgaaatcat tgctacactg 241aagaatggag ttcaaacatg tctaaaccca gattcagcag atgtgaagga actgattaaa 301aagtgggaga aacaggtcag ccaaaagaaa aagcaaaaga atgggaaaaa acatcaaaaa 361aagaaagttc tgaaagttcg aaaatctcaa cgttctcgtc aaaagaagac tacataagag 421accacttcac caataagtat tctgtgttaa aaatgttcta ttttaattat accgctatca 481ttccaaagga ggatggcata taatacaaag gcttattaat ttgactagaa aatttaaaac 541attactctga aattgtaact aaagttagaa agttgatttt aagaatccaa acgttaagaa 601ttgttaaagg ctatgattgt ctttgttctt ctaccaccca ccagttgaat ttcatcatgc 661ttaaggccat gattttagca atacccatgt ctacacagat gttcacccaa ccacatccca 721ctcacaacag ctgcctggaa gagcagccct aggcttccac gtactgcagc ctccagagag 781tatctgaggc acatgtcagc aagtcctaag cctgttagca tgctggtgag ccaagcagtt 841tgaaattgag ctggacctca ccaagctgct gtggccatca acctctgtat ttgaatcagc 901ctacaggcct cacacacaat gtgtctgaga gattcatgct gattgttatt gggtatcacc 961actggagatc accagtgtgt ggctttcaga gcctcctttc tggctttgga agccatgtga 1021ttccatcttg cccgctcagg ctgaccactt tatttctttt tgttcccctt tgcttcattc 1081aagtcagctc ttctccatcc taccacaatg cagtgccttt cttctctcca gtgcacctgt 1141catatgctct gatttatctg agtcaactcc tttctcatct tgtccccaac accccacaga 1201agtgctttct tctcccaatt catcctcact cagtccagct tagttcaagt cctgcctctt 1261aaataaacct ttttggacac acaaattatc ttaaaactcc tgtttcactt ggttcagtac 1321cacatgggtg aacactcaat ggttaactaa ttcttgggtg tttatcctat ctctccaacc 1381agattgtcag ctccttgagg gcaagagcca cagtatattt ccctgtttct tccacagtgc 1441ctaataatac tgtggaacta ggttttaata attttttaat tgatgttgtt atgggcagga 1501tggcaaccag accattgtct cagagcaggt gctggctctt tcctggctac tccatgttgg 1561ctagcctctg gtaacctctt acttattatc ttcaggacac tcactacagg gaccagggat 1621gatgcaacat ccttgtcttt ttatgacagg atgtttgctc agcttctcca acaataagaa 1681gcacgtggta aaacacttgc ggatattctg gactgttttt aaaaaatata cagtttaccg 1741aaaatcatat aatcttacaa tgaaaaggac tttatagatc agccagtgac caaccttttc 1801ccaaccatac aaaaattcct tttcccgaag gaaaagggct ttctcaataa gcctcagctt 1861tctaagatct aacaagatag ccaccgagat ccttatcgaa actcatttta ggcaaatatg 1921agttttattg tccgtttact tgtttcagag tttgtattgt gattatcaat taccacacca 1981tctcccatga agaaagggaa cggtgaagta ctaagcgcta gaggaagcag ccaagtcggt 2041tagtggaagc atgattggtg cccagttagc ctctgcagga tgtggaaacc tccttccagg 2101ggaggttcag tgaattgtgt aggagaggtt gtctgtggcc agaatttaaa cctatactca 2161ctttcccaaa ttgaatcact gctcacactg ctgatgattt agagtgctgt ccggtggaga 2221tcccacccga acgtcttatc taatcatgaa actccctagt tccttcatgt aacttccctg 2281aaaaatctaa gtgtttcata aatttgagag tctgtgaccc acttaccttg catctcacag 2341gtagacagta tataactaac aaccaaagac tacatattgt cactgacaca cacgttataa 2401tcatttatca tatatataca tacatgcata cactctcaaa gcaaataatt tttcacttca 2461aaacagtatt gacttgtata ccttgtaatt tgaaatattt tctttgttaa aatagaatgg 2521tatcaataaa tagaccatta atcagHuman CXCL9 Protein Sequence, Genbank #NP_002407.1 SEQ ID NO: 46MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTTHuman CXCL10 cDNA Sequence, Genbank #NM_001565.2; coding sequence: 71-367SEQ ID NO: 47 1gggggagaca ttcctcaatt gcttagacat attctgagcc tacagcagag gaacctccag 61tctcagcacc atgaatcaaa ctgccattct gatttgctgc cttatctttc tgactctaag 121tggcattcaa ggagtacctc tctctagaac tgtacgctgt acctgcatca gcattagtaa 181tcaacctgtt aatccaaggt ctttagaaaa acttgaaatt attcctgcaa gccaattttg 241tccacgtgtt gagatcattg ctacaatgaa aaagaagggt gagaagagat gtctgaatcc 301agaatcgaag gccatcaaga atttactgaa agcagttagc aaggaaaggt ctaaaagatc 361tccttaaaac cagaggggag caaaatcgat gcagtgcttc caaggatgga ccacacagag 421gctgcctctc ccatcacttc cctacatgga gtatatgtca agccataatt gttcttagtt 481tgcagttaca ctaaaaggtg accaatgatg gtcaccaaat cagctgctac tactcctgta 541ggaaggttaa tgttcatcat cctaagctat tcagtaataa ctctaccctg gcactataat 601gtaagctcta ctgaggtgct atgttcttag tggatgttct gaccctgctt caaatatttc 661cctcaccttt cccatcttcc aagggtacta aggaatcttt ctgctttggg gtttatcaga 721attctcagaa tctcaaataa ctaaaaggta tgcaatcaaa tctgcttttt aaagaatgct 781ctttacttca tggacttcca ctgccatcct cccaaggggc ccaaattctt tcagtggcta 841cctacataca attccaaaca catacaggaa ggtagaaata tctgaaaatg tatgtgtaag 901tattcttatt taatgaaaga ctgtacaaag tagaagtctt agatgtatat atttcctata 961ttgttttcag tgtacatgga ataacatgta attaagtact atgtatcaat gagtaacagg 1021aaaattttaa aaatacagat agatatatgc tctgcatgtt acataagata aatgtgctga 1081atggttttca aaataaaaat gaggtactct cctggaaata ttaagaaaga ctatctaaat 1141gttgaaagat caaaaggtta ataaagtaat tataactaaa aaaaHuman CXCL10 Protein Sequence, Genbank #NP_001556.2 SEQ ID NO: 48MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSPHuman IFN-alpha cDNA Sequence, Genbank #J00210.1; coding sequence: 221-790SEQ ID NO: 49 1aaaacaaaac atttgagaaa cacggctcta aactcatgta aagagtgcat gaaggaaagc 61aaaaacagaa atggaaagtg gcccagaagc attaagaaag tggaaatcag tatgttccct 121atttaaggca tttgcaggaa gcaaggcctt cagagaacct agagcccaag gttcagagtc 181acccatctca gcaagcccag aagtatctgc aatatctacg atggcctcgc cctttgcttt 241actgatggtc ctggtggtgc tcagctgcaa gtcaagctgc tctctgggct gtgatctccc 301tgagacccac agcctggata acaggaggac cttgatgctc ctggcacaaa tgagcagaat 361ctctccttcc tcctgtctga tggacagaca tgactttgga tttccccagg aggagtttga 421tggcaaccag ttccagaagg ctccagccat ctctgtcctc catgagctga tccagcagat 481cttcaacctc tttaccacaa aagattcatc tgctgcttgg gatgaggacc tcctagacaa 541attctgcacc gaactctacc agcagctgaa tgacttggaa gcctgtgtga tgcaggagga 601gagggtggga gaaactcccc tgatgaatgc ggactccatc ttggctgtga agaaatactt 661ccgaagaatc actctctatc tgacagagaa gaaatacagc ccttgtgcct gggaggttgt 721cagagcagaa atcatgagat ccctctcttt atcaacaaac ttgcaagaaa gattaaggag 781gaaggaataa catctggtcc aacatgaaaa caattcttat tgactcatac accaggtcac 841gctttcatga attctgtcat ttcaaagact ctcacccctg ctataactat gaccatgctg 901ataaactgat ttatctattt aaatatttat ttaactattc ataagattta aattattttt 961gttcatataa cgtcatgtgc acctttacac tgtggttagt gtaataaaac atgttcctta 1021tatttactca atccattatt ttgtgttgtt cattaaactt ttactatagg aacttcctgt 1081atgtgttcat tctttaatat gaaattccta gcctgactgt gcaacctgat tagagaataa 1141agggtatatt ttatttgctt atcattatta tatgtaagaHuman IFN-alpha Protein Sequence, Genbank #AAB59403.1 SEQ ID NO: 50MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKEHuman IFN-gamma IEF SSP 5111 cDNA Sequence, Genbank #L07633.1; coding sequence: 93-842SEQ ID NO: 51 1gcggagctgg gtgcgagcgc cctaccgctt tcgctttccc ttcgcggtgc ccactccact 61ccttgtgcgg cgctaggccc cccgtcccgg tcatggccat gctcagggtc cagcccgagg 121cccaagccaa ggtggatgtg tttcgtgaag acctctgtac caagacagag aacctgctcg 181ggagctattt ccccaagaag atttctgagc tggatgcatt tttaaaggag ccagctctca 241atgaagccaa cttgagcaat ctgaaggccc cattggacat cccagtgcct gatccagtca 301aggagaaaga gaaagaggag cggaagaaac agcaggagaa ggaagacaag gatgaaaaga 361agaaggggga ggatgaagac aaaggtcctc cctgtggccc agtgaactgc aatgaaaaga 421tcgtggtcct tctgcagcgc ttgaagcctg agatcaagga tgtcattgag cagctcaacc 481tggtcaccac ctggttgcag ctgcagatac ctcggattga ggatggtaac aattttggag 541tggctgtcca ggagaaggtg tttgagctga tgaccagcct ccacaccaag ctagaaggct 601tccacactca aatctctaag tatttctctg agcgtggtga tgcagtgact aaagcagcca 661agcagcccca tgtgggtgat tatcggcagc tggtgcacga gctggatgag gcagagtacc 721gggacatccg gctgatggtc atggagatcc gcaatgctta tgctgtgtta tatgacatca 781tcctgaagaa cttcgagaag ctcaagaagc ccaggggaga aacaaaggga atgatctatt 841gagagccctc tctcccattc tgtgatgagt acagcagaga ccttcctgct ttttactggg 901gactccagat tttccccaaa cttgcttctg ttgagatttt tccctcacct tgcctctcag 961gcacaataaa tatagttata ccactHuman IFN-gamma IEF SSP 5111 Protein Sequence, Genbank #AAA16521.1SEQ ID NO: 52MAMLRVQPEAQAKVDVFREDLCTKTENLLGSYFPKKISELDAFLKEPALNEANLSNLKAPLDIPVPDPVKEKEKEERKKQQEKEDKDEKKKGEDEDKGPPCGPVNCNEKIVVLLQRLKPEIKDVIEQLNLVTTWLQLQIPRIEDGNNFGVAVQEKVFELMTSLHTKLEGFHTQISKYFSERGDAVTKAAKQPHVGDYRQLVHELDEAEYRDIRLMVMEIRNAYAVLYDIILKNFEKLKKPRGETKGMIYHuman CCL-19 cDNA Sequence, Genbank #BC027968.1; coding sequence: 125-421SEQ ID NO: 53 1catcactcac accttgcatt tcacccctgc atcccagtcg ccctgcagcc tcacacagat 61cctgcacaca cccagacagc tggcgctcac acattcaccg ttggcctgcc tctgttcacc 121ctccatggcc ctgctactgg ccctcagcct gctggttctc tggacttccc cagccccaac 181tctgagtggc accaatgatg ctgaagactg ctgcctgtct gtgacccaga aacccatccc 241tgggtacatc gtgaggaact tccactacct tctcatcaag gatggctgca gggtgcctgc 301tgtagtgttc accacactga ggggccgcca gctctgtgca cccccagacc agccctgggt 361agaacgcatc atccagagac tgcagaggac ctcagccaag atgaagcgcc gcagcagtta 421acctatgacc gtgcagaggg agcccggagt ccgagtcaag cattgtgaat tattacctaa 481cctggggaac cgaggaccag aaggaaggac caggcttcca gctcctctgc accagacctg 541accagccagg acagggcctg gggtgtgtgt gagtgtgagt gtgagcgaga gggtgagtgt 601ggtcagagta aagctgctcc acccccagat tgcaatgcta ccaataaagc cgcctggtgt 661ttacaactaa aaaaaaaaaa aaaaaaaaaa aaaaHuman CCL-19 Protein Sequence, Genbank #AAH27968.1 SEQ ID NO: 54ALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSSHuman CCL-21 cDNA Sequence, Genbank #BC027918.1; coding sequence: 8-412SEQ ID NO: 55 1cacagacatg gctcagtcac tggctctgag cctccttatc ctggttctgg cctttggcat 61ccccaggacc caaggcagtg atggaggggc tcaggactgt tgcctcaagt acagccaaag 121gaagattccc gccaaggttg tccgcagcta ccggaagcag gaaccaagct taggctgctc 181catcccagct atcctgttct tgccccgcaa gcgctctcag gcagagctat gtgcagaccc 241aaaggagctc tgggtgcagc agctgatgca gcatctggac aagacaccat ccccacagaa 301accagcccag ggctgcagga aggacagggg ggcctccaag actggcaaga aaggaaaggg 361ctccaaaggc tgcaagagga ctgagcggtc acagacccct aaagggccat agcccagtga 421gcagcctgga gccctggaga ccccaccagc ctcaccagcg cttgaagcct gaacccaaga 481tgcaagaagg aggctatgct caggggccct ggagcagcca ccccatgctg gccttgccac 541actctttctc ctgctttaac caccccatct gcattcccag ctctaccctg catggctgag 601ctgcccacag caggccaggt ccagagagac cgaggaggga gagtctccca gggagcatga 661gaggaggcag caggactgtc cccttgaagg agaatcatca ggaccctgga cctgatacgg 721ctccccagta caccccacct cttccttgta aatatgattt atacctaact gaataaaaag 781ctgttctgtc ttcccaccca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 841aaaaaaaaaa aaaaaa Human CCL-21 Protein Sequence, Genbank #AAH27918.1SEQ ID NO: 56MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGPHuman TNF DNA Sequence, Genbank #NM_000594.2; coding sequence: 170-871SEQ ID NO: 57 1ctccctcagc aaggacagca gaggaccagc taagagggag agaagcaact acagaccccc 61cctgaaaaca accctcagac gccacatccc ctgacaagct gccaggcagg ttctcttcct 121ctcacatact gacccacggc tccaccctct ctcccctgga aaggacacca tgagcactga 181aagcatgatc cgggacgtgg agctggccga ggaggcgctc cccaagaaga caggggggcc 241ccagggctcc aggcggtgct tgttcctcag cctcttctcc ttcctgatcg tggcaggcgc 301caccacgctc ttctgcctgc tgcactttgg agtgatcggc ccccagaggg aagagttccc 361cagggacctc tctctaatca gccctctggc ccaggcagtc agatcatctt ctcgaacccc 421gagtgacaag cctgtagccc atgttgtagc aaaccctcaa gctgaggggc agctccagtg 481gctgaaccgc cgggccaatg ccctcctggc caatggcgtg gagctgagag ataaccagct 541ggtggtgcca tcagagggcc tgtacctcat ctactcccag gtcctcttca agggccaagg 601ctgcccctcc acccatgtgc tcctcaccca caccatcagc cgcatcgccg tctcctacca 661gaccaaggtc aacctcctct ctgccatcaa gagcccctgc cagagggaga ccccagaggg 721ggctgaggcc aagccctggt atgagcccat ctatctggga ggggtcttcc agctggagaa 781gggtgaccga ctcagcgctg agatcaatcg gcccgactat ctcgactttg ccgagtctgg 841gcaggtctac tttgggatca ttgccctgtg aggaggacga acatccaacc ttcccaaacg 901cctcccctgc cccaatccct ttattacccc ctccttcaga caccctcaac ctcttctggc 961tcaaaaagag aattgggggc ttagggtcgg aacccaagct tagaacttta agcaacaaga 1021ccaccacttc gaaacctggg attcaggaat gtgtggcctg cacagtgaag tgctggcaac 1081cactaagaat tcaaactggg gcctccagaa ctcactgggg cctacagctt tgatccctga 1141catctggaat ctggagacca gggagccttt ggttctggcc agaatgctgc aggacttgag 1201aagacctcac ctagaaattg acacaagtgg accttaggcc ttcctctctc cagatgtttc 1261cagacttcct tgagacacgg agcccagccc tccccatgga gccagctccc tctatttatg 1321tttgcacttg tgattattta ttatttattt attatttatt tatttacaga tgaatgtatt 1381tatttgggag accggggtat cctgggggac ccaatgtagg agctgccttg gctcagacat 1441gttttccgtg aaaacggagc tgaacaatag gctgttccca tgtagccccc tggcctctgt 1501gccttctttt gattatgttt tttaaaatat ttatctgatt aagttgtcta aacaatgctg 1561atttggtgac caactgtcac tcattgctga gcctctgctc cccaggggag ttgtgtctgt 1621aatcgcccta ctattcagtg gcgagaaata aagtttgctt agaaaagaaHuman TNF Protein Sequence, Genbank #NP_000585.2 SEQ ID NO: 58MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALHuman IL-27 cDNA Sequence, Genbank #BC062422.1; coding sequence: 27-758SEQ ID NO: 59 1ggggaccaaa gaggctgggc cccgccatgg gccagacggc aggcgacctt ggctggcggc 61tcagcctgtt gctgcttccc ttgctcctgg ttcaagctgg tgtctgggga ttcccaaggc 121ccccagggag gccccagctg agcctgcagg agctgcggag ggagttcaca gtcagcctgc 181atctcgccag gaagctgctc gccgaggttc ggggccaggc ccaccgcttt gcggaatctc 241acctgccagg agtgaacctg tacctcctgc ccctgggaga gcagctccct gatgtttccc 301tgaccttcca ggcctggcgc cgcctctctg acccggagcg tctctgcttc atctccacca 361cgcttcagcc cttccatgcc ctgctgggag ggctggggac ccagggccgc tggaccaaca 421tggagaggat gcagctgtgg gccatgaggc tggacctccg cgatctgcag cggcacctcc 481gcttccaggt gctggctgca ggattcaacc tcccggagga ggaggaggag gaagaggagg 541aggaggagga ggagaggaag gggctgctcc caggggcact gggcagcgcc ttacagggcc 601cggcccaggt gtcctggccc cagctcctct ccacctaccg cctgctgcac tccttggagc 661tcgtcttatc tcgggccgtg cgggagttgc tgctgctgtc caaggctggg cactcagtct 721ggcccttggg gttcccaaca ttgagccccc agccctgatc ggtggcttct tagccccctg 781ccccccaccc tttagaactt taggactgga gtcttggcat cagggcagcc ttcgcatcat 841cagccttgga caagggaggg ctcttccagc cccctgcccc aggccctacc cagtaactga 901aagcccctct ggtcctcgcc agctatttat ttcttggata tttatttatt gtttagggag 961atgatggttt atttattgtc ttggggcccg atggtcctcc tcgggccaag cccccatgct 1021gggtgcccaa taaagcactc tcatccataa aaaaaaaaaa aaaaaaaaaa aaaaaaaHuman IL-27 Protein Sequence, Genbank #AAH62422.1 SEQ ID NO: 60MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREFTVSLHLARKLLAEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWFLGFPTLSPQPmCherry sequence SEQ ID NO: 61ATGGTGAGCA AGGGCGAGGA GGATAACATG GCCATCATCA AGGAGTTCAT GCGCTTCAAG GTGCACATGGAGGGCTCCGT GAACGGCCAC GAGTTCGAGA TCGAGGGCGA GGGCGAGGGC CGCCCCTACG AGGGCACCCAGACCGCCAAG CTGAAGGTGA CCAAGGGTGG CCCCCTGCCC TTCGCCTGGG ACATCCTGTC CCCTCAGTTCATGTACGGCT CCAAGGCCTA CGTGAAGCAC CCCGCCGACA TCCCCGACTA CTTGAAGCTG TCCTTCCCCGAGGGCTTCAA GTGGGAGCGC GTGATGAACT TCGAGGACGG CGGCGTGGTG ACCGTGACCC AGGACTCCTCCCTGCAGGAC GGCGAGTTCA TCTACAAGGT GAAGCTGCGC GGCACCAACT TCCCCTCCGA CGGCCCCGTAATGCAGAAGA AGACCATGGG CTGGGAGGCC TCCTCCGAGC GGATGTACCC CGAGGACGGC GCCCTGAAGGGCGAGATCAA GCAGAGGCTG AAGCTGAAGG ACGGCGGCCA CTACGACGCT GAGGTCAAGA CCACCTACAAGGCCAAGAAG CCCGTGCAGC TGCCCGGCGC CTACAACGTC AACATCAAGT TGGACATCAC CTCCCACAACGAGGACTACA CCATCGTGGA ACAGTACGAA CGCGCCGAGG GCCGCCACTC CACCGGCGGC ATGGACGAGCTGTACAAGTA A Mouse CCL-21-mCherry-mINT fusion DNA sequence SEQ ID NO: 62ATGGCTCAGATGATGACTCTGAGCCTCCTTAGCCTGGTCCTGGCTCTCTGCATCCCCTGGACCCAAGGCAGTGATGGAGGGGGTCAGGACTGCTGCCTTAAGTACAGCCAGAAGAAAATTCCCTACAGTATTGTCCGAGGCTATAGGAAGCAAGAACCAAGTTTAGGCTGTCCCATCCCGGCAATCCTGTTCTCACCCCGGAAGCACTCTAAGCCTGAGCTATGTGCAAACCCTGAGGAAGGCTGGGTGCAGAACCTGATGCGCCGCCTGGACCAGCCTCCAGCCCCAGGGAAACAAAGCCCCGGCTGCAGGAAGAACCGGGGAACCTCTAAGTCTGGAAAGAAAGGAAAGGGCTCCAAGGGCTGCAAGAGAACTGAACAGACACAGCCCTCAAGAGGAAGATCCATGGTGAGCA AGGGCGAGGA GGATAACATG GCCATCATCA AGGAGTTCAT GCGCTTCAAG GTGCACATGGAGGGCTCCGT GAACGGCCAC GAGTTCGAGA TCGAGGGCGA GGGCGAGGGC CGCCCCTACG AGGGCACCCAGACCGCCAAG CTGAAGGTGA CCAAGGGTGG CCCCCTGCCC TTCGCCTGGG ACATCCTGTC CCCTCAGTTCATGTACGGCT CCAAGGCCTA CGTGAAGCAC CCCGCCGACA TCCCCGACTA CTTGAAGCTG TCCTTCCCCGAGGGCTTCAA GTGGGAGCGC GTGATGAACT TCGAGGACGG CGGCGTGGTG ACCGTGACCC AGGACTCCTCCCTGCAGGAC GGCGAGTTCA TCTACAAGGT GAAGCTGCGC GGCACCAACT TCCCCTCCGA CGGCCCCGTAATGCAGAAGA AGACCATGGG CTGGGAGGCC TCCTCCGAGC GGATGTACCC CGAGGACGGC GCCCTGAAGGGCGAGATCAA GCAGAGGCTG AAGCTGAAGG ACGGCGGCCA CTACGACGCT GAGGTCAAGA CCACCTACAAGGCCAAGAAG CCCGTGCAGC TGCCCGGCGC CTACAACGTC AACATCAAGT TGGACATCAC CTCCCACAACGAGGACTACA CCATCGTGGA ACAGTACGAA CGCGCCGAGG GCCGCCACTC CACCGGCGGC ATGGACGAGCTGTACAAGTA Atgcacacaacactg gcaggatgct gtgccttgga cagaactcct cagtctacag acagaggatggcttctggaa acttacacca gaactgggac ttatattaaatcttaataca aatggtttgc acagctttct taaacaaaaa ggcattcaat ctctaggtgt aaaaggaagagaatgtctcc tggacctaat tgccacaatg ctggtactac agtttattcg caccaggttg gaaaaagagggaatagtgtt caaatcactg atgaaaatgg atgacccttc tatttccagg aatattccct gggcttttgaggcaataaag caagcaagtg aatgggtaag aagaactgaaggacagtacc catctatctg cccacggcttgaactgggga acgactggga ctctgccacc aagcagttgc tgggactcca gcccataagc actgtgtcccctcttcatag agtcctccat tacagtcaag gctaa Mouse CCL21-forward primerSEQ ID NO: 63 GCGCGGATCCCCATGGCTCAGATGATG Mouse CCL-21-reverse primerSEQ ID NO: 64 GCGCAGATCTTCCTCTTGAGGGCTGTGTCTG Human CCL21 primerSEQ ID NO: 65 CCCCACTAGTCCAGTTCTCAGTCACTGGCTCTG Human CCL21 primerSEQ ID NO: 66 CCCCGCTAGCTGGCCCTTTAGGGGTCTGTG Human CCL21 primerSEQ ID NO: 67 CCCCGCTAGCTGCACACAACACTGGCAGGA Human CCL21 primerSEQ ID NO: 68 GGGGCTCGAGTTAGCCTTGACTGTAATGGAHuman mINT protein sequence (residues 1473-1724 of human VPARP protein sequence)SEQ ID NO: 69Ala Asn Leu Arg Leu Pro Met Ala Ser Ala Leu Pro Glu Ala Leu CysSer Gln Ser Arg Thr Thr Pro Val Asp Leu Cys Leu Leu Glu GluSer Val Gly Ser Leu Glu Gly Ser Arg Cys Pro Val Phe Ala PheGln Ser Ser Asp Thr Glu Ser Asp Glu Leu Ser Glu Val Leu GlnAsp Ser Cys Phe Leu Gln Ile Lys Cys Asp Thr Lys Asp Asp SerIle Pro Cys Phe Leu Glu Leu Lys Glu Glu Asp Glu Ile Val CysThr Gln His Trp Gln Asp Ala Val Pro Trp Thr Glu Leu Leu SerLeu Gln Thr Glu Asp Gly Phe Trp Lys Leu Thr Pro Glu Leu GlyLeu Ile Leu Asn Leu Asn Thr Asn Gly Leu His Ser Phe Leu LysGln Lys Gly Ile Gln Ser Leu Gly Val Lys Gly Arg Glu Cys LeuLeu Asp Leu Ile Ala Thr Met Leu Val Leu Gln Phe Ile Arg ThrArg Leu Glu Lys Glu Gly Ile Val Phe Lys Ser Leu Met Lys MetAsp Asp Pro Ser Ile Ser Arg Asn Ile Pro Trp Ala Phe Glu AlaIle Lys Gln Ala Ser Glu Trp Val Arg Arg Thr Glu Gly Gln TyrPro Ser Ile Cys Pro Arg Leu Glu Leu Gly Asn Asp Trp Asp SerAla Thr Lys Gln Leu Leu Gly Leu Gln Pro Ile Ser Thr Val SerPro Leu His Arg Val Leu His Tyr Ser Gln Gly

REFERENCES CITED

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1. A composition comprising a vault complex comprising a chemokinefusion protein comprising a chemokine (C-C motif) ligand 21 (CCL-21)consisting of the amino acid sequence of SEQ ID NO:2 (human) and a majorvault protein interaction domain (mINT) consisting of the amino acidsequence of SEQ ID NO:8 (human).
 2. A composition comprising a vaultcomplex comprising a fusion protein comprising a cytokine and a vaulttargeting domain.
 3. The composition of claim 2, wherein the cytokine isa chemokine.
 4. The composition of claim 2, wherein the cytokine is acysteine-cysteine (CC) chemokine.
 5. The composition of claim 2, whereinthe cytokine is a CCL-21 chemokine.
 6. The composition of claim 2,wherein the cytokine comprises the amino acid sequence of SEQ ID NO:2(human).
 7. The composition of claim 2, wherein the cytokine consists ofthe amino acid sequence of SEQ ID NO:2 (human).
 8. The composition ofclaim 2, wherein the cytokine comprises the amino acid sequence of SEQID NO:1 (mouse).
 9. The composition of claims 2-8, wherein the vaulttargeting domain is a vault interaction domain from a vault polyADP-ribose polymerase (VPARP).
 10. The composition of claims 2-8,wherein the vault targeting domain is a major vault protein interaction(mINT) domain.
 11. The composition of claims 2-8, wherein the vaulttargeting domain comprises the amino acid sequence of SEQ ID NO:8(human).
 12. The composition of claims 2-8, wherein the vault targetingdomain consists of the amino acid sequence of SEQ ID NO:8 (human). 13.The composition of claims 2-8, wherein the vault targeting domaincomprises the amino acid sequence of SEQ ID NO:9 (mouse).
 14. Thecomposition of claims 2-13, wherein the vault complex comprises MVP. 15.The composition of claims 2-14, wherein the fusion protein comprises theamino acid sequence of SEQ ID NO:13 (human).
 16. The composition ofclaims 2-14, wherein the fusion protein consists of the amino acidsequence of SEQ ID NO:13 (human).
 17. The composition of claims 2-14,wherein the fusion protein comprises the amino acid sequence of SEQ IDNO:12 (mouse).
 18. The composition of claims 2-17, further comprising avault poly ADP-ribose polymerase (VPARP), a telomerase vault associatedprotein 1 (TEP1), or an untranslated RNA molecule (vRNA).
 19. Thecomposition of claims 2-18, wherein the fusion protein further comprisesa fluorescent protein.
 20. The composition of claims 2-18, wherein thefluorescent protein is a mCherry fluorescent protein.
 21. An isolatednucleic acid encoding a chemokine fusion protein comprising a cytokineencoding sequence and a mINT encoding sequence.
 22. The isolated nucleicacid of claim 21, wherein the mINT encoding sequence comprises thenucleic acid sequence of SEQ ID NO:7 (human).
 23. The isolated nucleicacid of claim 21, wherein the mINT encoding sequence consists of thenucleic acid sequence of SEQ ID NO:7 (human).
 24. The isolated nucleicacid of claim 21, wherein the mINT encoding sequence comprises thenucleic acid sequence of SEQ ID NO:6 (mouse).
 25. The isolated nucleicacid of claim 21, wherein the cytokine encoding sequence consists of thenucleic acid sequence of SEQ ID NO:5 (human) and the mINT encodingsequence consists of the nucleic acid sequence of SEQ ID NO:7 (human).26. The isolated nucleic acid of claim 21, wherein the cytokine encodingsequence consists of the nucleic acid sequence of SEQ ID NO:3 (mouse)and the mINT encoding sequence consists of the nucleic acid sequence ofSEQ ID NO:6 (mouse).
 27. The isolated nucleic acid of claim 21, whereinthe cytokine fusion protein comprises the nucleic acid sequence of SEQID NO:11 (human).
 28. The isolated nucleic acid of claim 21, wherein thecytokine fusion protein consists of the nucleic acid sequence of SEQ IDNO:11 (human).
 29. The isolated nucleic acid of claim 21, wherein thecytokine fusion protein comprises the nucleic acid sequence of SEQ IDNO:10 (mouse).
 30. The isolated nucleic acid of claim 21, wherein thecytokine fusion protein comprises the nucleic acid sequence of SEQ IDNO:62 (mouse).
 31. A vector comprising the isolated nucleic acid ofclaims 21-30.
 32. The vector of claim 30, wherein the vector is abaculovirus expression vector.
 33. A cell comprising the nucleic acid ofclaims 21-30 or the vector of claims 31-32.
 34. A method of delivering acytokine to a cell, comprising introducing the composition of claims2-20 to the cell.
 35. The method of claim 34, wherein the composition isintroduced into the extracellular environment surrounding the cell. 36.A method for stimulating an immune response in a cell, the methodcomprising contacting the cell with the composition of claims 2-20. 37.The method of claim 36, wherein the cell is a human cell.
 38. The methodof claim 36 or 37, wherein the immune response induces migration of Tcells and dendritic cells.
 39. The method of claims 36-38, whereincontacting the cell with the composition of claim 2 increases T cellmigration to the cell by at least 5% compared to administration ofCCL-21 cytokine alone.
 40. A method for stimulating an immune responsein a subject, the method comprising administering the composition ofclaims 2-20 to the subject.
 41. The method of claim 40, wherein thesubject is a human.
 42. A method of treating or managing cancer in asubject in need of treatment or management of cancer, comprisingadministering to a subject a therapeutically effective amount of thecomposition of claims 2-20.
 43. The method of claim 42, whereinadministering comprises intra-tumoral injection of the composition to atumor in the subject.
 44. The method of claims 42-43, wherein the canceris lung cancer.
 45. The method of claims 42-44, wherein administeringreduces tumor volume.
 46. The method of claims 42-45, whereinadministering reduces tumor growth.
 47. The method of claims 42-46,wherein administering increases interleukin-2 (IL-2) expression.
 48. Themethod of claim 42-47, wherein the subject is a mammal.
 49. The methodof claims 42-47, wherein the subject is a human.
 50. A method ofpreparing the composition of claims 2-20 comprising a) mixing a fusionprotein comprising a cytokine fused to a mINT generated in Sf9 cellswith a rat MVP generated in Sf9 cells to generate a mixture; b)incubating the mixture for a sufficient period of time to allowpackaging of the fusion protein inside of vault complexes, therebygenerating the composition of claims 2-20.
 51. A method of preparing thecomposition of claims 2-20 comprising a) mixing a fusion proteincomprising a cytokine fused to a mINT generated in insect larvae cellswith a rat MVP generated in insect larvae cells to generate a mixture;b) incubating the mixture for a sufficient period of time to allowpackaging of the fusion protein inside of vault complexes, therebygenerating the composition of claims 2-20.
 52. A method of preparing thecomposition of claims 2-20 comprising a) mixing a fusion proteincomprising a cytokine fused to a mINT generated in Sf9 cells or insectlarvae cells with a human MVP generated in Sf9 cells or insect larvaecells to generate a mixture; b) incubating the mixture for a sufficientperiod of time to allow packaging of the fusion protein inside of vaultcomplexes, thereby generating the composition of claims 2-20.